IMMUNITY 


AND 


SPECIFIC    THERAPY 


BY 

W.  D'ESTE  EMERY,   M.D.,  B.Sc.  LOND. 

CLINICAL  PATHOLOGIST  TO   KING'S  COLLEGE  HOSPITAL  AND  PATHOLOGIST  TO  THE   CHILDREN'S 
*       HOSPITAL,   PADDINGTON  GREEN  ;    FORMERLY  ASSISTANT  BACTERIOLOGIST  TO  THE  ROYAL 
COLLEGES  OF  PHYSICIANS  AND  SURGEONS,  AND  SOMETIME  LECTURER  ON  PATHOLOGY 
AND  BACTERIOLOGY  IN  THE  UNIVERSITY  OF  BIRMINGHAM 


WITH  ILLUSTRATIONS 


OF   THE 

UNIVERSITY 

OF 


PAUL  B.  HOEBER 

69,    EAST    59T.H    STREET 

NEW  YORK 

1909 


PRINTED  IN  ENGLAND 


PREFACE 

IN  writing  this  book  I  have  attempted  to  give  a  connected  and 
symmetrical  outline  of  the  chief  facts  definitely  known  with  regard 
to  the  method  in  which  the  body  protects  itself  against  infections, 
and  of  their  applications  in  the  diagnosis,  prevention,  and  treat- 
ment of  disease.  It  is  not  written  in  support  of  the  views  of  any 
particular  school  of  thought,  and,  when  dealing  with  subjects  still 
under  discussion,  I  have  tried  to  give  a  fair  and  impartial,  though 
necessarily  succinct,  account  of  each  of  the  rival  theories.  The 
factors  in  many  of  the  problems  of  immunity  are  so  complex, 
and  our  knowledge  of  the  subject  grows  and  alters  so  rapidly, 
that  it  is  quite  impossible  to  deal  with  it  dogmatically  at  the 
present  time.  I  have  kept  in  view,  as  far  as  possible,  the  re- 
quirements of  the  physician  and  surgeon  who  may  require  an 
epitome  of  the  theoretical  basis  of  the  modern  methods  of  diagnosis 
and  treatment,  now  assuming  so  much  importance,  and  of  the 
student  who  desires  a  general  survey  of  the  subject  before  com- 
mencing more  advanced  studies. 

My  best  thanks  are  due  to  Mr.  H.  K.  Lewis  for  the  ready  and 
courteous  way  in  which  he  has  acceded  to  all  my  suggestions  and 
requirements ;  to  Drs.  Whitfield  and  Briscoe,  from  whom  I  have 
received  some  valuable  suggestions;  and  to  Professor  Herbert 
Jackson,  of  King's  College,  for  kindly  reading  the  sections  dealing 
with  the  more  purely  chemical  and  physical  questions  and  for 
much  useful  information  connected  therewith.  I  have  also  to 
thank  Sir  Almroth  Wright  and  Drs.  R.  W.  Allen,  Eyre,  and 
Bolduan;  Messrs.  Macmillan  and  Co.,  Kegan  Paul  and  Co.,  and 
the  proprietors  of  the  Lavcet,  British  Medical  Journal,  and  the  St. 
Bartholomew's  Hospital  Journal  for  permission  to  use  illustrations 
from  their  publications. 


CONTENTS 

CHAPTER  PACE 

GLOSSARY                   .                  .  '               .                                                       .  ix 

I.    INTRODUCTORY    AND    GENERAL       .                  ...  I 

II.    ON    THE    NATURE    OF    TOXINS          .  .  .  -37 

III.    THE    PHENOMENA    OF    ANTITOXIN    FORMATION        .                  .  60 

IV.    INTERREACTIONS    OF    TOXIN    AND    ANTITOXIN          .                  .  69 

V.    THE    ORIGIN    OF    ANTITOXIN— THE    SIDE-CHAIN    THEORY.  Q2 

VI.    IMMUNITY    TO    TOXINS        .                  .                  .                  .                  •  IT5 

VII.    BACTERIOLYSIS    AND    ALLIED    PHENOMENA                 .                  .  139 

VIII.    THE    AGGLUTININS                 .                  .                  .                                    .  204 

IX.    THE    PRECIPITINS                                      ....  226 

X.    PHAGOCYTOSIS        .                  .                  .                  .                                -.    .  238 

xi.  "REACTIONS"  AND  SIMILAR  PHENOMENA            .             .  300 

XII.    COLLOIDAL    THEORY    OF    ANTIBODIES            .                  .                  .  319 

XIII.  ON    IMMUNITY    TO    BACTERIA             ....  33! 

XIV.  PRACTICAL  APPLICATIONS             ....  358 

BIBLIOGRAPHY      .  .  .  .  .  .421 

LIST  OF  AUTHORITIES     .            .            .            .            .  439 

INDEX       .            .            .            .            .            .            .  443 


ERRATA 

Page      9,  line  5  from  bottom,  omit  "  to  "  after  "  -cytes." 
14,  line  13,  for  "  rather  of  "  read  "  than  with." 
36,  line  22,  for  "  of  read  "to." 
,,       45,  line  29,  for  "  antitoxin  "  read  "  toxin." 

48,  line  14.  for  "became"  read  "become";    line  25,  for  "  united 

with"  read  "injured." 

,,       52,  line  10.  for  "  supernatural  "  read  "  supernatant.  " 
,,       55,  line  4  from  bottom,  for  "  properties  "  read  "  effects." 

57,  line  i,  for  "is"  read  "  are  "  ;  bottom  line,  insert  "  upon  "  after 

"  toxins,"  and  for  "  defends  "  read  "  depends." 
,,       70.  line  6.  for  "  haemoglobin  "  read  "  haemolysin." 
,,       78,  lines  17  and  19,   for  "  c.c.  "  read  "parts";   and  line  18,  for 

"  16-6  c.c.  "  read  "  i6'6  parts." 
79,  top  line,  for  "antitoxin  "  read  "  toxin." 
90,  line  22,  for  "  toxin  "  read  "  antitoxin." 
,,       91,  line  18,  for  "  toxic  "  read  "  neutral"  ;  line  26,  omit  "  as. " 

93,  line  22,  for  "injection"  read  "infection." 
,,     106,  line  26,  for  "it"  read  "  the  toxin  " 

,,     107,  line  21,  for  "  to  tetanus  antitoxin  "  read  "of  tetanus  toxin." 
,,     no,  line  9,  for  "  antitoxin"  read  "  toxin." 
122,  line  3,  for  "  toxin  "  read  "  antitoxin." 

128,  line  20,  for  "  leucocytes  "  read  "  bacteria  ingested." 

129,  line  3  from  bottom,  for  "  toxin"  read  "  antitoxin." 
152,  line  7,  for  "joined  "  read  "  formed." 

171,  line  10,  for  "  rabbit"  read  "  goat." 

192,  line  30.  for  "  nephrotoxin  "  read  "  hepatotoxin." 

220,  line  7  from  bottom,  for  "  they  "  read  "it." 

252,  line  12,  for  "leucocytes  "  read  "bacteria." 

256,  line  15,  for  "complement"  read  "amboceptor." 

284,  line  13,  for  "  opsonix  "  read  "  opsonic." 

287,  line  8,  for  "which  in"  read  "in  which." 

290,  line  19,  for  "  bacteria  can  "  read  "  leucocytes  can   ' 

296,  line  8,  for  "  bacteria  "  read  "  leucocytes." 

307,  line  12.  for  "  local  "  read  •'  general." 

310,  line  6  from  bottom,  for  "  y  "  read  "a." 

323,  line  i,  for  "or"  read  "on";  line  28,  for  "  complement  "  read 

"  amboceptor." 
3-15    line  13,  should  reaJ,  "those  which  hava  no  defensive  layer,  or 

which  have  numerous  receptors  "  etc. 
348,  line  14,  for  "  bacteria"  read  "  leucocytes." 
351,  line  14,  for  "installations  "  read  "instillations." 
355,  last  line,  for  "research  "  read  "defence." 
360,  line  3,  for  "  able  "  read  •'  unable." 
365,  line  29.  for  '"  -stable  ' '  read  "  labile. ' ' 
378,  line  8  from  bottom, read  "are  accompanied  by  but  the  slightest," 

etc. 

386,  line  21.  for  'benefits  "  read  '"benefit." 
407,  line  10  from  bottom,  for  '•  rise  "  read  <;use." 
419,  line  25,  for  "heated  "  read  "beaten." 


GLOSSARY 

Active  immunity.  Immunity  due  to  an  active  struggle  against  some  infective 
material,  vaccine,  or  toxin. 

Addiment.     See  Alexin  or  Complement. 

Agglutinin.  A  specific  antibody  which  brings  about  agglutination  —  i.e., 
causes  the  bacteria,  cells,  etc.,  for  which  it  is  specific,  to  collect  into 
clumps.  Non-specific  substances  (acids,  etc.)  have  a  similar  action,  but 
are  not  properly  termed  agglutinins. 

Agglutinogen.  The  antigen  of  agglutinin— i.e.,  the  substance  which,  when 
injected  into  a  suitable  animal,  leads  to  the  formation  of  agglutinin. 

Agglutinoid,  A  modification  of  agglutinin  which  has  retained  the  power  of 
uniting  with  the  specific  bacteria,  etc.,  but  has  lost  that  of  causing  them 
to  clump. 

Aggressin  (aggredior,  I  attack).  A  substance  secreted  by  bacteria  and  possess- 
ing the  power  of  inhibiting  phagocytosis  of  the  organism  producing  it. 

Alexin  (dX&jw,  I  ward  off).  A  defensive  substance  having  an  injurious  effect 
on  bacteria,  and  occurring  in  the  serum  of  normal  and  immune  animals. 
It  is  analogous  in  many  respects  to  the  bacterial  toxins,  and,  like  them, 
easily  destroyed  by  heat,  chemical  agents,  etc.  It  is  probably  identical 
with  complement,  q.v.  (For  other  synonyms,  see  p  143.) 

Amboceptor  (ambo,  both,  and  capio,  I  take).  A  specific  antibody  produced 
by  the  injection  of  bacteria,  red  corpuscles,  cells,  etc.,  and  exerting,  with 
the  help  of  alexin  or  complement,  a  solvent  action  on  these  substances. 
The  term  is  Ehrlich's,  and  its  use  should  involve  the  acceptance  of  his 
theory  of  its  action.  (For  synonyms,  see  p.  142.) 

Anaphylaxis  (a  or  dm,  privative,  and  0uXa<r(rw,  1  guard).  The  opposite  of 
prophylaxis — i.e.,  a  condition  in  which  the  susceptibility  of  the  animal 
(especially  to  toxins,  serums,  etc.)  is  abnormally  increased.  Practically 
identical  with  hypersensitiveness. 

Antibody.  A  substance  formed  by  the  injection  into  an  animal  of  a  substance 
(its  antigen)  not  normally  found  in  the  juices  of  that  animal  (and  probably 
in  all  cases  of  proteid  constitution,  or  closely  allied  thereto),  which  unites 
with  its  antigen  and  modifies  it  in  some  way. 

Antiferment,  or  antienzyme.     An  antibody  to  a  ferment  or  enzyme. 

Antigen  (dvTi,  against,  and  yiyvu.  I  produce).  A  substance  which,  when 
injected  into  a  suitable  animal,  has  the  power  of  leading  to  the  produc- 
tion of  an  antibody.  In  most,  if  not  in  all,  cases  it  is  proteid  in  nature. 

Antitoxin.  An  antibody  to  a  toxin — i.e.,  a  specific  substance  formed  by  an 
animal  in  consequence  of  the  presence  in  its  tissues  or  juices  of  a  given 
toxin,  which  the  antitoxin  thus  produced  has  the  power  of  neutralizing. 

Arthus'  phenomenon.  A  form  of  hypersensitiveness  to  serum,  where  in  a 
sensitized  animal  local  lesions  (gargrene,  abscesses,  etc.)  develop  in  a 
region  where  serum  is  injected,  and  the  animal  may  become  cachectic 
and  die. 

Atrepsy  (d,  privative,  and  rptyw,  I  nourish).  A  condition  in  which  an  infective 
agent  or  collection  of  malignant  cells  dies  in  the  animal  body  owing  to 
its  being  unable  to  obtain  suitable  nourishment  :  a  conception  intro- 
duced by  Ehrlich  to  explain  certain  forms  of  immunity — e.g.,  to  malignant 
growths. 

Attenuation.  The  change  which  an  organism  undergoes  whereby  it  becomes 
less  virulent. 


X  GLOSSARY 

Autohsemolysin.  A  substance  which  has  the  power  of  dissolving  the  red  cor- 
puscles of  the  individual  which  produces  it ;  an  autochthonous  ambo- 
ceptor  or  immune  body  to  an  animal's  own  red  corpuscles. 

Bacteriolysis.  The  phenomenon  of  solution  of  bacteria,  more  especially  by 
the  action  of  specific  antibodies,  aided  by  alexin  or  complement. 

Bacteriotropin,  or  bacteriotropic  substance  (Tptiru,  I  turn).  A  substance 
(usually  a  specific  antibody)  which  has  the  property  of  uniting  with 
bacteria,  and  in  someway  altering  their  properties,  usually  rendering  them 
more  suitable  for  phagocytosis. 

Bordet's  phenomenon. — The  absorption  of  alexin  or  complement  which  is 
brought  about  by  means  of  a  cell  or  bacterium  combined  with  amboceptor 
or  immune  body,  apart  altogether  from  the  alexin  which  is  necessary  for 
the  action  ;  the  complete  removal  of  all  alexin  from  a  fluid  by  means  of 
a  cell-immune-body  compound. 

Chemotaxis  (rdfa,  an  arrangement).  The  attraction  or  repulsion  of  leucocytes, 
bacteria,  etc.,  by  substances  in  solution  in  the  fluid  containing  the  cells 
in  question. 

Complement  (compleo,  I  fill  up).  A  synonym  for  alexin,  q.v.  The  term  was 
introduced  by  Ehrlich,  since  on  his  theory  this  substance  unites  with  the 
amboceptor  which  has  already  united  with  the  bacterium,  etc.,  and  thus 
completes  the  conditions  necessary  for  solution. 

Complementoid.  A  modification  of  complement  which  possesses  the  combin- 
ing powers  of  that  substance,  but  which  has  lost  its  active  solvent 
properties. 

Cytase.  Metchnikoffs  term  for  the  digestive  (proteolytic)  enzyme  secreted 
by  leucocytes  ;  a  synonym  for  alexin  or  complement. 

Cytolysin.  An  antibody  (immune  body  or  amboceptor)  to  a  cell,  having  the 
power  of  sensitizing  that  cell  so  that  it  is  completely  or  partially  dissolved 
on  the  addition  of  alexin. 

Cytolysis.  The  solution — usually  partial — of  cells  by  means  of  an  antibody 
(immune  body  or  amboceptor)  and  alexin. 

Cytotoxin.  A  substance  acting  as  a  cellular  toxin,  especially  an  antibody  and 
alexin  ;  practically  identical  with  a  cytolysin. 

Danysz  effect.  The  decrease  in  the  neutralizing  effect  of  antitoxin  which  is 
manifested  when  the  toxin  is  added  in  portions,  with  an  interval  between 
each,  rather  than  all  at  once. 

Desmon  (5e<r/<i6s,  a  bond).      A  synonym  for  immune  body  or  amboceptor. 

Deviation  of  complement.  The  phenomenon  in  which  the  solvent  effect  of 
immune  body  on  cells  or  bacteria  (in  presence  of  alexin)  diminishes  as  an 
excess  of  the  antibody  is  added  ;  also  known  as  the  Neisser-Wechsberg 
phenomenon. 

Dominant  complement.  Where  (on  Ehrlich's  theory)  two  or  more  different 
complements  unite  with  a  complex  molecule  of  amboceptor  which  has 
united  with  a  bacterium,  corpuscle,  or  cell,  that  which  has  the  more 
potent  action  is  known  as  the  dominant.  Its  effect  may  be  produced 
without  the  action  of  the  other  complements,  or  the  necessary  amount 
may  be  smaller. 

Ehrlich's  phenomenon.  The  fact  that  the  difference  between  the  amount  of 
toxin  exactly  neutralized  by  one  unit  of  antitoxin  and  the  amount  which 
(added  to  one  unit  of  antitoxin)  just  leaves  one  lethal  unit  free  is  greater 
than  one  lethal  dose  of  simple  toxin. 

Endocomplement.  A  complement  contained  within  a  red  corpuscle,  probably 
in  all  cases  lecithin  or  an  allied  substance. 

Endotoxin.  A  bacterial  toxin  contained  within  the  substance  of  the  bticterium, 
and  not  liberated  except  when  the  cell  is  destroyed. 

Ergophore  group  (tpyov,  work,  and  0^/>ar,  I  bear).  The  part  of  a  molecule  of 
antigen  or  antibody  on  which  the  specific  properties  of  the  substance 
depends  (toxophore,  zymophore,  agglutinophore,  etc.),  in  distinction  from 
the  haptophore  or  combining  part  of  the  molecule. 


GLOSSARY  XI 

Exotoxin.  A  soluble  bacterial  toxin  which  is  excreted  by  the  bacterium  into 
the  surrounding  fluid  during  the  life  of  the  organism. 

Fixation  of  complement.     A  synonym  for  Bordet's  phenomenon,  q.v. 
Fixator.     A  synonym  for  immune  body  or  amboceptor. 

Gastrotoxin.     A  cytotoxin  or  cytolysin  acting  on  the  cells  of  the  mucous 

membrane  of  the  stomach. 
Gengou's  reaction.     The  removal  of  all  alexin  or  complement  from  a  fluid  by 

means  of  a  compound  of  a  precipitin  and  its  antigen  ;  analogous  with 

Bordet's  phenomenon,  except  that  in  this  case  the  reacting  antigen  is  a 

soluble  substance.    The  two  are  often  grouped  together  as  the  Bordet- 

Gengou  reaction. 
Group  reaction.     A  reaction  with  an  antibody  (usually  an  agglutinin)  which 

is  common  to  several  species  of  bacteria,  forming  a  well-defined  group — 

e.g. ,  the  coli  group,  or  the  pasteurelloses. 

Haemagglutinin.     A  substance  which  agglutinates  red  corpuscles. 

Hsemolysin.  A  substance  which  dissolves  red  blood-corpuscles,  or  at  least 
releases  the  haemoglobin  which  they  contain.  The  term  is  used  mainly 
for  an  antibody  having,  in  conjunction  with  alexin,  a  solvent  action  of 
this  nature. 

Haptin.  A  portion  of  a  molecule  of  protoplasm  having  combining  affinities 
for  food  molecules,  and  forming  an  antibody  when  shed  (v.  Receptor). 

Haptophore  group,  or  Radicle  (CCTTTW,  I  fasten).  That  portion  of  a  substance 
(whether  antigen  or  antibody)  which  has  the  power  of  entering  into  com- 
bination with  its  appropriate  antibody  or  antigen,  as  the  case  may  be. 
Thus  a  molecule  of  toxin  is  supposed  to  contain  a  group  of  atoms  which 
can  combine  with  a  cell  or  molecule  of  antitoxin,  and  a  second  which  can 
then  exert  a  toxic  action.  The  former  is  known  as  the  haptophore 
group. 

Immune  body  (immunis,  exempt  from  public  service).  A  specific  antibody, 
produced  by  the  injection  of  bacteria  or  other  cells,  and  having  the 
power  of  altering  these  substances  in  such  a  way  as  to  render  them  com- 
pletely or  partially  soluble  on  the  addition  of  alexin.  It  is  the  same  as 
amboceptor,  but  the  term  implies  no  theory  and  is  generally  preferable. 
Synonyms  :  substance  sensibilatrice,  desmon,  preparator,  copula,  etc. 

Incitor  constituent  of  serum.  A  substance  which  aids  phagocytosis,  espe- 
cially thermostable  opsonin. 

Isoagglutinins.  An  agglutinin  which, '  occurring  in  the  serum  of  a  certain 
animal,  will  agglutinate  the  red  corpuscles  of  other  animals  of  that 
species,  but  not  those  of  the  individual  which  produces  it. 

Isohsemolysin.  An  immune  body  or  amboceptor  which,  occurring  in  the 
serum  of  a  certain  animal,  dissolves  (in  conjunction  with  alexin)  the  red 
corpuscles  of  other  animals  of  that  species,  but  not  those  of  the  individual 
which  produces  it. 

Koch's  phenomenon.  The  tuberculin  reaction,  or  rise  of  temperature  and 
sudden  exacerbation  of  the  local  lesions  occurring  in  a  tuberculous  animal 
after  injection  of  a  culture  of  tubercle  bacilli,  living  or  dead,  tuberculin, 
or  other  specific  tuberculous  material. 

Lactoserum.     A  serum  containing  a  precipitin  for  milk  proteids. 

Leucotoxin.  An  antibody  (immune  body  or  amboceptor)  which,  in  conjunc- 
tion with  alexin,  exerts  a  toxic  influence  on  leucocytes. 

Lysis  (Xims,  a  loosening).  The  solution  of  cells,  bacteria,  etc.,  mostly  by 
means  of  antibodies  or  other  protective  substances. 

LO  dose  of  toxin.  The  amount  which  is  exactly  neutralized  by  one  unit  of 
antitoxin. 

L+  dose  of  toxin.  The  amount  which,  added  to  one  unit  of  antitoxin,  behaves 
just  like  one  lethal  dose  of  toxin,  bringing  about  a  fatal  result  in  test 
animals  within  the  time-limit  fixed.  The  fact  that  the  L_|_  dose -the  L0 
dose  is  greater  than  one  lethal  dose  constitutes  the  Ehrlich  phenomenon. 


Xll  GLOSSARY 

Macrocytase.  In  Metchnikoff's  phraseology,  the  digestive  enzyme  secreted  by 
the  large  mononuclear  leucocytes,  and  having  a  special  action  on  cells 
rather  than  on  bacteria ;  really  a  synonym  for  alexin,  especially  for  one 
acting  on  cells  or  red  corpuscles. 

Macrophage.  Metchnikoff's  term  for  a  large  phagocyte  which,  according  to 
him,  is  especially  adapted  to  the  ingestion  of  cells  or  corpuscles  rather 
than  of  bacteria  They  may  be  large  lymphocytes,  large  hyaline  cells, 
endothelial  or  other  tissue  cells. 

Microcytase.  The  digestive  enzyme  of  Metchnikoff's  microcytes  or  poly- 
nuclear  leucocytes  ;  supposed  to  have  a  special  action  on  bacteria. 
Practically  identical  with  alexin. 

Microphage.  A  small  leucocyte  supposed  by  Metchnikoff  to  be  specially 
active  against  bacteria,  and  to  have  little  or  no  phagocytic  action  on  cells 
or  corpuscles.  They  are  polynuclear  leucocytes. 

Negative  phase.  The  sudden  diminution  in  the  amount  of  an  antibody  (and 
possibly  of  other  defensive  substances)  in  the  blood  which  follows 
immediately  on  the  injection  of  an  antigen. 

Neisser-Wechsberg  phenomenon.     Deviation  of  the  complement,  q.v. 

Nephrotoxin.     A  cytotoxin  specific  for  renal  cells. 

-ogen.  A  suffix  usually  employed  to  denote  an  antigen  in  relation  to  its  anti- 
body— e.g.,  agglutinogen,  the  substance  which  on  injection  into  an 
animal  leads  to  the  production  of  agglutinin.  Also  used  for  a  preliminary 
non-active  form  of  an  active  substance — e.g.,  opsoninogen,  a  substance 
which  under  certain  conditions  becomes  opsonin. 

-oid  (eldos,  a  figure  or  appearance).  A  suffix  denoting  a  secondary  modifica- 
tion of  an  active  substance  in  which  it  appears  to  retain  its  power  of 
entering  into  combination  with  its  antibody  or  antigen,  but  has  lost  its 
specific  activity  ;  a  molecule  of  antigen  or  antibody  which  has  lost  its 
ergophore,  but  retained  its  toxophore,  group—  e.g.,  complementoid  or 
toxoid,  q.v. 

Opsonin  (opsono — I  cater  for,  I  prepare  for  food.  Derived  from  &\}/ov,  cooked 
meat,  a  sauce  or  relish).  A  substance  or  combination  of  substances  of 
whatever  nature  which  has  the  power  of  combining  with  a  bacterium, 
cell,  or  other  substance,  and  rendering  it  more  easily  ingested  by  a 
leucocyte  or  other  phagocyte. 

Passive  immunity.  Immunity  due  to  the  injection  of  serum  from  an  animal 
which  has  acquired  immunity  to  a  toxin  or  infective  agent. 

Pfeiffer's  phenomenon.  The  classical  Pfeiffer's  phenomenon  consists  in  the 
globular  transformation,  loss  of  staining  reaction,  and  finally  complete 
disappearance  of  cholera  vibrios,  when  introduced  into  the  peritoneal 
cavity  of  an  immunized  guinea-pig,  or  into  that  of  a  normal  one  if 
immune  serum  be  also  injected.  Also  applied  to  the  similar,  but  usually 
less  complete,  destruction  of  other  bacteria  under  similar  conditions,  or 
to  bacteriolysis  in  general. 

Phytotoxin.  A  poisonous  substance  formed  by  one  of  the  higher  plants,  but 
otherwise  closely  resembling  a  bacterial  toxin,  more  especially  in  its 
power  to  give  rise  to  the  production  of  an  antitoxin  on  injection — e.g., 
ricin,  abrin. 

Polyceptor.  Amboceptor  which  possesses  several  haptophore  groups  capable 
of  anchoring  several  molecules  of  different  sorts  of  complement,  the 
most  important  of  which  is  termed  the  dominant  (Ehrlich). 

Polyvalent  serum.  A  serum  containing  antibodies  against  several  strains  of 
the  same  species  of  bacteria— e.g.,  streptococci. 

Polyvalent  vaccine.  A  vaccine  composed  of  the  dead  bodies  of  several  strains 
of  the  same  bacterial  species.  A  vaccine  composed  of  more  than  one 
species  of  organism  is  termed  a  mixed  vaccine. 

Positive  phase.  The  period  during  which  the  amount  of  antibody  or  other 
protective  body  in  the  serum  is  increased  owing  to  the  injection  of  an 
antigen.  In  general  terms  it  corresponds  to  the  period  of  exalted  im- 


GLOSSARY  Xlll 

munity  due  to  vaccination,  injection  of  toxin,  etc.,  and  is  very  variable 
in  duration. 

Precipitin.  An  antibody  to  a  soluble  form  of  proteid,  having  the  power  of 
precipitating  or  coagulating  that  proteid  by  a  process  of  clumping  its 
molecules. 

Precipitogen.  The  antigen  to  a  given  precipitin.  Thus  when  a  serum  is 
injected  into  an  animal  numberless  substances  are  introduced,  a  certain 
number  of  which  only  give  rise  to  the  formation  of  precipitin,  and  are 
called  precipitogens.  Also  called  precipitable  substance. 

Precipitogenoid.  Heated  precipitable  substance,  which  has  retained  its 
power  of  combining  with  precipitin,  but  no  longer  forms  a  precipitate 
after  doing  so. 

Precipitoid.  Precipitin  which  has  lost  its  active  or  ergophore,  but  retained  its 
combining  or  haptophore,  group  ;  the  latter  has  also  increased  in 
affinity  for  precipitable  substance.  The  name  is  also  applied  to  pre- 
cipitogenoid. 

Predisposition.  The  opposite  of  immunity  ;  the  state  of  an  animal,  in  virtue 
of  which  it  is  readily  infected  with  a  given  agent. 

Preparator.     Metchnikorf's  term  for  immune  body  or  amboceptor. 

Prophylaxis.  Any  process  by  which  the  vulnerability  of  an  animal  by  an 
infective  agent  or  toxin  is  diminished  or  removed  ;  a  process  for  the 
induction  of  immunity,  more  especially  in  its  practical  application  to  the 
prevention  of  disease. 

Prostatotoxin.     Axcytolysin  for  the  cells  of  the  prostate. 

Pro-zone.  In  constructing  a  curve  indicating  the  action  of  an  antibody  at 
different  dilutions,  it  sometimes  happens  that  stronger  solutions  have 
less  effect  than  more  dilute  ones.  The  region  of  the  curve  in  which  this 
inhibition  of  the  action  is  brought  about  by  an  excess  of  the  active  sub- 
stance is  termed  the  pro-zone.  It  occurs  with  substances  other  than 
antibodies.  Also  called  zone  of  inhibition. 

Receptor.  In  Ehrlich's  side-chain  theory  a  part  of  a  living  molecule  of  pro- 
toplasm which  has  the  power  of  attracting  and  combining  with  a  molecule 
of  food  proteid  (or  of  toxin,  etc.)  from  the  fluid  with  which  it  is  bathed, 
and  of  building  it  up  into  the  whole  molecule,  and  thus  utilizing  it  as 
nourishment,  to  aid  which  process  it  may  also  seize  one  or  more 
molecules  of  complement.  When  shed  into  the  blood  these  receptors 
constitute  antibodies. 

1.  Simple  (e.g.,  those  constituting  antitoxin).     In  the  antibodies  formed 
by  this  group  we  can  only  distinguish  one  group  o'f  atoms — a  haptophore 
group  having  the  power  of  combining  with  the  specific  antigen   (e.g., 
toxin),  and  preventing  its  subsequent  union  with  a  living  cell,  thus  render- 
ing it  inert. 

2.  Complex  (e.g.,  agglutinin),  in  which  we  can  recognize  two  separate 
properties,  presumably  situate  in  different  groups  of  atoms  :  (a)  a  hapto- 
phore, combining  group,  as  above ;    and    (b)   an   ergophore  group,  on 
which   the  activity  depends,  and  which   may  be   destroyed   whilst   (a) 
remains  intact. 

3.  Compound  (e.g.,  amboceptor,  on  Ehrlich's  theory).     In  them  there 
are  t-uv  or  more  haptophore  groups,  one  of  which  combines  with  the 
antigen,  the  others  with  one  or  more  molecules  of  complement. 

Sensitization  of  bacteria,  corpuscles,  etc.  The  addition  of  immune  body,  so 
that  the  objects  are  prepared  or  sensitized  to  the  action  of  alexin. 

Side-chain  theory.  The  theory  (Ehrlich's)  which  accounts  for  the  develop- 
ment of  antibodies  by  supposing  that  the  receptors  (q.v.)  which  combine 
with  the  specific  antigen  may,  under  certain  circumstances,  be  produced 
in  excess  and  cast  off  into  the  surrounding  fluid  ;  these  receptors,  retain- 
ing their  power  of  combining  with  antigen,  constitute  the  antibodies  in 
question.  A  brilliant  conception,  which  has  been  the  cause  of  enormous 
advance  in  our  knowledge  of  problems  connected  with  immunity. 


XIV  GLOSSARY 

Smith's  (Theobald)  phenomenon.  The  acquisition  of  hypersensitiveness  to 
serum  and  other  proteid  substances  (normally  inert)  which  occurs  in 
some  animals  as  a  result  of  minute  doses  of  these  substances,  and  leads 
to  rapid  death,  with  acute  symptoms,  when  a  second  injection  is  given. 

Specificity  (species,  an  image).  A  direct  relation  of  cause  and  effect  between 
two  substances  (such  as  diphtheria  toxin  and  its  antitoxin,  the  latter  being 
only  produced  by,  and  acting  only  on,  the  former),  or  between  a  substance 
and  a  phenomenon  (such  as  the  tuberculin  reaction,  produced  only  by 
tuberculous  products  in  a  tuberculous  animal).  The  specific  products  of 
a  micro-organism  are  those  produced  only  by  that  organism,  so  that  their 
recognition  is  proof  of  its  presence.  In  the  same  way  a  specific  disease 
is  one  produced  only  by  a  certain  bacterium  (such  as  diphtheria  or 
anthrax),  and  not  by  several  organisms  (such  as  suppuration  or  actino- 
mycosis) . 

Spermotoxin.     A  cytolysin  to  spermatozoa. 

Stimulin.  A  substance  having  the  power  of  stimulating  the  action  of  the 
leucocytes  (more  especially  in  regard  to  phagocytosis)  by  a  direct  action 
on  the  leucocyte  itself.  The  existence  of  these  substances  is  doubtful, 
most  of  the  phenomena  supposed  to  be  caused  by  them  being  due  (a)  to 
the  action  of  opsonins,  and  (b)  to  substances  which  have  a  positive 
chemotactic  action,  attracting  leucocytes  to  the  region. 

Syncytiotoxin.     A  cytolysin  acting  on  the  cells  of  the  placenta. 

Thermolabile.  Easily  destroyed  by  heat.  In  general  thermolabile  substances 
are  destroyed,  completely  or  partially,  by  an  exposure  to  55°  C.  for  half 
an  hour  or  to  60°  C.  for  10  minutes. 

Thermostable.     The  opposite  to  thermolabile,  q.v. 

Thyrotoxin.     A  cytolysin  acting  on  the  cells  of  the  thyroid  gland. 

Toxin  (ro&Kbv  <j>dpiu.a.Koi> ,  the  drug  with  which  poisoned  arrows  were  anointed. 
TO^OV,  a  bow).  The  specific  poison  on  which  the  pathogenic  activity  of  a 
micro-organism  depends.  The  fact  of  its  being  specific  excludes  simple 
chemical  substances  which  may  also  exert  a  toxic  action. 

Toxoid.  A  secondary  modification  of  a  toxin  which  has  lost  its  power  of 
producing  toxic  symptoms,  but  retained  that  of  combining  with  antitoxin 
or  susceptible  cells  ;  or,  in  Ehrlich's  terminology,  one  that  has  lost  its 
toxophore,  but  retained  its  haptophore,  group. 

Toxone.  A  specific  substance  of  feeble  toxicity  and  slight  affinity  for  anti- 
toxin which  is  supposed  to  be  produced  by  certain  bacteria,  notably  that 
of  diphtheria,  in  which  case  it  is  believed  to  be  the  cause  of  paralysis. 
Unlike  toxoid,  it  is  a  primary  product.  Its  existence  is  doubted,  and  the 
effects  attributed  to  minute  amounts  of  toxin  by  some  authors. 

Toxophore  group.  The  portion  of  a  molecule  of  toxin  on  which  the  toxic 
activity  depends,  the  destruction  of  which  converts  the  molecule  into  one 
of  toxoid. 

Trichotoxin.     A  specific  cytotoxin  for  ciliated  epithelium. 

Vaccination.  The  production  of  active  immunity  by  some  process  less 
severe  than  the  induction  of  an  ordinary  attack  of  the  disease  in 
question. 

Vaccine.  A  substance  (usually  a  dead  culture  or  living  culture  of  mitigated 
virulence)  the  injection  of  which  leads  to  the  production  of  active  im- 
munity with  less  risk  than  that  which  accompanies  an  ordinary  attack  of 
the  disease. 

Virulence  (virus,  a  poison).  The  property  or  properties  of  an  organism  in 
virtue  of  which  it  is  able  to  give  rise  to  disease  in  animals  or  to  produce 
a  powerful  toxin. 

Zootoxin.  A  poisonous  substance  of  animal  origin  which  resembles  in  other 
respects  (and  especially  in  that  it  can  give  rise  to  the  production  of  an 
antitoxin)  the  bacterial  toxins — e.g.,  snake  venom,  eel  serum. 

Zymophore  group  (&M,  leaven).  The  portion  of  an  enzyme  or  enzyme-like 
substance  on  which  the  specific  properties  depend,  in  contradistinction  to 
the  combining  or  haptophore  portion. 


OF   THE 

UNIVERSITY 

OF 

>R^ 


IMMUNITY  AND  SPECIFIC 
THERAPY 

CHAPTER  I 
INTRODUCTORY  AND  GENERAL 

IMMUNITY  is  the  power  which  certain  living  organisms  possess  of 
resisting  influences  which  are  deleterious  to  others.  In  its  widest 
form  it  includes  the  power  of  resisting  poisons,  adverse  physical 
influences,  and  diseases  of  all  kinds.  Thus,  many  men  can  and 
do  acquire  some  degree  of  immunity  against  nicotine,  alcohol,  and 
other  poisons  ;  some  bacteria  are  immune  to  temperatures  which 
are  quickly  fatal  to  others  ;  and  some  individuals  and  races  have 
a  very  real  immunity  to  gout  and  other  metabolic  diseases  to 
which  their  less  fortunate  brethren  are  more  prone.  In  any 
complete  discussion  of  the  subject  these  forms  of  immunity  would 
require  some  consideration,  but  in  what  follows  we  shall,  in  the 
main,  limit  ourselves  to  the  investigation  of  immunity  against 
the  diseases  of  bacterial  origin.  In  doing  so  we  must  not  be 
thought  to  consider  the  other  diseases — metabolic  and  what  not 
— as  being  unimportant.  The  very  reverse  is  the  case,  and  the 
subject  which  calls  most  urgently  for  research  at  the  present  day 
is  the  nature  and  mechanism  of  immunity  against  malignant 
tumours,  and  of  this  we  have  recently  acquired  a  little  know- 
ledge. But  the  diseases  other  than  those  of  bacterial  origin  will 
not  be  dealt  with,  for  the  simple  reason  that  our  knowledge  of 
their  intimate  causes  is  still  unknown,  and  until  they  are  dis- 
covered, and  until  the  physiological  disturbances  of  the  economy 
which  occur  in  these  diseases  are  more  fully  known,  the  nature 
of  the  corresponding  immunity  is  obviously  extremely  difficult 
of  study.  The  bacterial  diseases  are  quite  different,  for  here 


2  INTRODUCTION 

the  causes  are  fully  known ;  the  diseases  themselves  can  be 
reproduced  (in  most  cases)  at  pleasure,  and  the  physiological  dis- 
turbances which  take  place  are  fairly  well  investigated.  There 
are,  of  course,  gaps,  and  those  not  inconsiderable,  in  our  know- 
ledge ;  but,  on  the  whole,  the  nature  of  these  diseases  is  nearly  as 
well  ascertained  as  the  present  state  of  normal  physiology  will 
allow.  Further,  we  can  not  only  reproduce  the  diseases,  but  we 
can  reproduce  in  most  cases  any  degree  of  immunity  to  them 
which  we  may  require  for  purposes  of  protection  or  research,  and 
we  can  investigate  the  differences  between  the  cells  and  fluids  of 
the  immunized  person  or  animal  and  the  corresponding  parts  of  a 
normal  organism,  and  we  can  attempt  to  correlate  them  with  the 
production  of  the  immune  state.  We  have,  therefore,  a  very 
large  amount  of  information  on  the  subject,  and  although  this 
information  is  at  present  incomplete,  we  have  already  obtained 
results  of  the  highest  practical  and  theoretical  importance  ;  and 
the  value  of  these  results  leads  us  to  believe  with  confidence  that 
our  methods  are  right,  that  we  are  on  the  right  track,  and  that  a 
solution  of  the  problems  that  have  at  present  baffled  research  will 
come  in  the  near  future. 

As  denned  above,  immunity  is  a  function  of  all  living  material, 
and  one  of  the  highest  importance.  Biologists  have  compiled 
lists  of  the  essential  properties  of  living  protoplasm — nutrition, 
reproduction,  and  the  like — but  have  not  realized  that  immunity 
to  bacterial  action  is  the  first  necessity  for  continued  life. 
Consider  for  a  moment  a  small  water  animal — say  a  hydra — 
occurring  in  water  which  naturally  contains  saprophytic  bacteria. 
Whilst  the  animal  lives  these  organisms  do  not  affect  its  proto- 
plasm in  any  way,  the  latter  being  immune  to  their  action  ;  but 
on  the  animal's  death  rapid  putrefaction  occurs,  and  in  a  few 
hours  its  protoplasm  is  broken  down  by  bacterial  action:  the 
immunity  has  ceased.  Immunity  to  putrefactive  bacteria  is 
therefore  a  condition  of  life  in  the  lower  animals.  But  the  same 
is  true  in  every  respect  for  those  of  a  higher  grade,  man  included. 
From  the  moment  of  birth  we  are  surrounded  with  air  containing 
bacteria  which  are  not  pathogenic  in  the  ordinary  sense,  but 
which  only  fail  to  be  so  because  of  the  inherent  power  of  immunity 
to  saprophytic  bacteria,  which  is  a  fundamental  property  of  all 
living  material.  Apart  from  this,  the  organisms  present  in  the 
air,  alimentary  canal,  skin,  etc.,  would  flourish  as  rapidly  as  they 
do  in  a  corpse,  and  life  would  only  be  possible  for  a  few  hours, 


INTRODUCTORY  AND   GENERAL  3 

or  perhaps  minutes.  Readers  of  one  of  Mr.  Wells's  ingenious 
romances  may  perhaps  remember  how  the  strange  beasts  from 
Mars  which  invaded  this  planet  died  rapidly,  being  evolved  in  a 
region  in  which  there  were  no  bacteria,  and  in  which  this  power 
of  resisting  their  action  had  not  been  developed.  The  example  is 
a  striking  one,  and  is  strictly  scientific,  though  we  may  wonder 
how  the  rotation  of  nitrogen,  in  which  bacteria  play  so  essential  a 
part,  is  brought  about  in  Mars  ;  for  this  process  of  the  breaking 
down  of  dead  proteids  by  bacterial  action,  and  the  preparation  of 
its  nitrogen  for  use  in  plants,  is  essential  for  continued  life  on  the 
planet.  Without  decomposition  all  the  combined  nitrogen  of  the 
world  would  soon  become  locked  up  in  the  dead  bodies  of  animals  ; 
plants  would  starve  and  die,  and  animals  (which  are  all  dependent, 
directly  or  indirectly,  on  plant  nitrogen)  would  likewise  become  ex- 
tinct. It  is  a  most  marvellous  natural  phenomenon  that  these  putre- 
factive bacteria  should  be  found  wherever  life  occurs,  and  wherever 
their  aid  may  be  required  to  deal  with  the  protoplasm  when  dead, 
and  that  this  same  protoplasm  should  have  acquired  such  potency 
in  resisting  their  attacks  whilst  still  alive.  Absence  of  bacteria 
or  absence  of  immunity  are  alike  incompatible  with  animal  life. 

Considerations  of  this  nature  lead  us  to  a  short  discussion  of 
the  difference  between  the  pathogenic  and  non-pathogenic  bacteria, 
and  we  find  that  there  is,  theoretically,  none.  Any  bacterium 
will  produce  disease  if  it  grows  in  the  tissues  of  the  living  body, 
and  all  bacteria1  will  do  so  if  the  necessary  degree  and  form  of 
immunity  is  not  present.  A  pathogenic  organism  is  one  which 
can  grow  in  the  living  tissues,  and  it  can  do  so  only  because  those 
mechanisms  of  immunity  which  are  sufficient  in  the  case  of  the 
saprophytic  bacteria  are  powerless  to  resist  it ;  but  in  most  cases, 
as  we  shall  show,  a  higher  degree  of  immunity  can  be  produced 
artificially,  and  the  microbe  in  question  then  becomes  non-patho- 
genic to  that  particular  animal.  So,  too,  with  the  bacteria 
ordinarily  regarded  as  non-pathogenic.  Under  certain  circum- 
stances, some  of  which  are  known  and  some  still  unknown,  the 
resistance  of  the  body  or  of  a  part  of  it  may  be  broken  down  to 
such  an  extent  that  these  organisms  may  gain  access,  flourish, 
and  give  rise  to  disease.  Thus,  B.  proteus  may  give  rise  to 
phlebitis,  growing  in  the  thrombosed  vein,  and  giving  off  toxins 
which  have  an  injurious  action  on  the  tissues. 

1  Bacteria  growing  only  at  very  high  or  very  low  temperatures,  or  on  media 
very  poor  in  nitrogen,  perhaps  excepted. 

I — 2 


4  INTRODUCTION — PATHOGENICITY 

As  a  matter  of  high  theory,  therefore,  there  is  no  fundamental 
distinction  between  pathogenic  and  non-pathogenic  bacteria,  and 
we  can  imagine  circumstances  in  which  the  tissues  are  vulnerable 
to  attack  by  almost  any  microbic  species.  Practically,  however, 
we  shall  consider  an  organism  as  pathogenic  when  the  immunity 
of  the  animal  which  it  attacks  is  not  so  perfectly  developed  that 
its  presence  in  the  tissues  is  but  transient  and  unaccompanied  by 
any  noticeable  ill-effects,  but  in  which  there  is  a  balanced  contest 
of  longer  or  shorter  duration  between  the  injurious  powers  of  the 
microbe  and  the  defensive  mechanism  of  the  host,  accompanied 
by  more  or  less  injury  to  the  tissues  and  disturbances  of  the 
physiological  economy  of  the  latter,  and  resulting  either  in  the 
death  of  the  invader  or  of  the  patient.  All  grades  occur.  In 
most  staphylococcic  infections  the  chances  are  enormously  on 
the  side  of  the  host,  and  the  immunity  is  sufficiently  high  to 
localize  the  process  before  it  has  gone  far.  In  typhoid  fever  the 
natural  immunity  and  the  pathogenic  power  of  the  organisms  are 
more  nicely  matched  ;  the  contest  between  them  is  of  long  duration 
and  doubtful  issue.  And  in  some  forms  of  human  disease,  but 
more  especially  in  artificial  infections  of  animals  with  highly 
virulent  cultures,  the  power  of  immunity  seems  almost  nothing, 
the  bacterium  growing  apparently  unchecked  and  death  occurring 
within  a  few  hours.  We  say  that  these  organisms  have  different 
degrees  of  pathogenicity,  but  it  would  be  equally  correct  to  say 
that  there  are  different  degrees  of  resistance  against  them,  since  an 
organism  that  is  highly  virulent  towards  one  animal  species  may 
be  quite  harmless  to  another,  so  that  pathogenicity  is  not  an 
inherent  property  of  certain  bacteria. 

Thus  far  we  have  considered  the  resistance  of  the  host  as  if  it 
were  fixed  and  definite,  but  this  is  not  the  case.  It  has  been 
known  from  time  immemorial  that  certain  diseases — especially 
those  due  to  infection — are  followed  by  a  greater  or  smaller  degree 
of  immunity,  so  that  a  second  attack  is  unlikely — at  any  rate,  for 
some  time.  Smallpox,  scarlet  fever,  and  measles  are  amongst  the 
most  striking  examples,  and  in  them  the  protection  given  by  the 
disease  is  in  most  instances  absolute  and  lifelong.  This  is  known 
as  acquired  immunity,  and  we  shall  enunciate  it  as  a  law  that  all 
recovery  from  infective  disease  is  due  to,  and  followed  by,  some 
degree  of  acquired  immunity,  though  this  may  be  slight,  transient, 
and  perhaps  local. 

Take,  for  example,  a  case  of  pneumonia,  a  disease  which  may 


INTRODUCTORY   AND    GENERAL  5 

occur  repeatedly  and  at  short  intervals  in  the  same  person. 
Pneumococci  are  widely  distributed,  and  are  almost  universally 
present  in  the  mouth ;  the  necessary  exciting  cause,  therefore,  is 
always  at  hand.  Under  ordinary  circumstances  the  power  of 
resistance  is  sufficient  to  ward  off  the  infection,  but  when  this 
barrier  of  immunity  is  broken  down  by  certain  adverse  circum- 
stances— by  excessive  fatigue  or  starvation,  by  cold,  or  by  an  over- 
dose of  alcohol  or  other  poison — the  pneumococcus  gains  access 
to  the  tissues,  and  infection1  occurs.  The  balanced  contest 
spoken  of  above  then  takes  place.  The  pneumococcus  grows  in 
'  the  lungs  and  blood  and  produces  a  toxin,  which  tends  to  reduce 
the  general  health  and  the  resistance  of  the  body  still  further;  and 
looking  at  the  problem  only  from  this  side,  it  would  appear  that 
the  process  would  go  on  until  all  the  immunity  was  broken  down, 
and  the  pneumococcus  could  flourish  unchecked.  This,  indeed, 
might  perhaps  happen  did  not  death  supervene  and  bring  with  it 
conditions  unfavourable  for  the  growth  of  this  organism.  But  all 
this  time  the  tissues  of  the  host  have  been  reacting,  and  (in  non- 
fatal  cases)  sooner  or  later  a  condition  is  brought  about  in  which 
the  noxious  power  of  the  coccus  and  the  immunity  of  the  patient 
are  exactly  level,  so  that  the  disease  neither  advances  nor  retro- 
cedes ;  and  the  process  goes  still  farther,  and  the  patient  develops 
such  a  degree  of  resistance  as  will  not  only  render  him  immune  to 
the  spread  of  the  infection,  but  will  suffice  to  sterilize  his  tissues 
of  the  pneumococci  which  have  already  gained  access.  In  other 
words,  there  has  been  an  acquisition  of  immunity;  the  patient  has 
become  immune  to  the  pneumococcus,  and  it  is  this,  and  this 
only,  which  has  brought  about  the  cure  of  the  disease. 

This  process  may  be  represented  very  diagrammatically,  as 
shown  on  p.  6. 

The  line  ag  represents  the  degree  of  immunity  to  the  organism 
in  question,  the  pneumococcus.  At  b  some  event  takes  place 
(e.g.,  exposure  to  cold)  by  which  the  resistance  is  lowered  to  such 
a  degree  that  infection  can  occur.  This  takes  place  at  c,  with  the 
result  that  the  immunity  falls  still  farther.  At  this  time  the 
bacteria  begin  to  flourish  in  the  tissues  in  increasing  numbers. 
This  is  represented  by  the  ascending  line  i.  The  immunity  falls 
and  bacterial  action  increases  until  a  certain  point  is  reached, 

1  I  have  elsewhere  defined  infection  as  the  access  of  living,  virulent, 
pathogenic  bacteria  to  a  region  whence  their  toxins  may  act  on  the  tissues  of 
the  body  (Rose  and  Carless's  "  Surgery,"  sixth  edition  et  seq.,  chap.  i.). 


6  RECOVERY   FROM    DISEASE 

when  the  reserve  forces  of  the  patient  have  been  brought  into 
action,  with  the  result  that  the  immunity  rises  (from  d  to  e). 
Somewhere  during  this  rise  (not  necessarily  or  probably  at  its 
commencement)  the  contest  turns  in  favour  of  the  host ;  the  bacteria 
are  rapidly  destroyed,  and  the  disease  is  cured.  Usually,  but  not 
necessarily,  there  is  a  rise  to  a  level  higher  than  the  previous 
normal  one  (e  to/),  of  longer  or  shorter  duration,  and  then  a  rever- 
sion to  the  normal  g.  If  exposure  to  cold  again  takes  place,  a 
fresh  infection  may  now  occur. 

Now  it  must  be  emphasized  that  natural  recovery  from  disease 
only  takes  place  in  virtue  of  an  acquisition  of  immunity  to  the 
infecting  agent,  and  in  no  other  way ;  and,  further,  that,  except  in 
a  few  instances,  medical  treatment  simply  aims  in  aiding  this 
phenomenon.  If  we  exclude  the  various  sera  and  vaccines,  there 


k 


FIG.  i. 

are  but  two  therapeutic  agents  which  have  a  direct  curative  effect 
— mercury  in  syphilis  and  quinine  in  malaria.1  In  these  diseases 
the  physician  can  apply  a  direct  remedy,  but  in  other  cases  the 
aim  and  object  of  treatment  is  to  support  the  patient's  strength 
until  the  natural  development  of  acquired  immunity  takes  place, 
and  in  some  cases  to  aid  this  development  by  certain  empirical 
means.  It  is  found  that  all  agents  which  tend  to  improve  the 
general  vitality  and  facilitate  the  performance  of  the  normal  physio- 
logical processes  have  this  action  ;  hence  the  importance  of  suitable 
food  in  amounts  and  at  intervals  suited  to  the  patient's  com- 
plaint, of  fresh  air  at  a  proper  temperature,  of  the  removal  of 
pain,  and  other  symptoms  which  tend  to  impair  the  patient's 

1  Arsenic  and  some  other  drugs  in  the  treatment  of  various  protozoal 
infections  (trypanosomiasis,  etc.)  may  also  be  included.  It  is  interesting  to 
notice  that  all  the  diseases  directly  combated  by  simple  means  are  protozoal 
in  origin. 


INTRODUCTORY  AND   GENERAL  7 

strength.  These  agents  are  all-important  in  medical  treatment 9 
but  in  themselves  they  are  useless,  and  they  only  act  by  hastening 
the  evolution  of  the  immunity,  without  which  the  disease  must 
necessarily  progress  to  a  fatal  issue.  This  is  well  seen  in  the  few 
diseases  in  which  the  development  of  immunity,  in  face  of  a  natural 
infection,  is  but  slight,  or  perhaps  altogether  absent,  such  as 
leprosy  or  hydrophobia.  Here  ordinary  medical  treatment  is 
powerless,  and  all  our  hopes  for  the  future  are  concerned  with  the 
discovery  of  a  direct  specific  remedy. 

It  is  this  connection  between  immunity  and  recovery  that 
renders  the  subject  so  important  to  the  physician,  and  the  neglect 
with  which  its  study  is  treated  by  the  general  members  of  the  pro- 
fession a  matter  of  such  profound  regret.  In  our  medical  educa- 
tion at  the  present  day  we  pay,  and  rightly,  much  attention  to  the 
study  of  physiology,  for  without  a  knowledge  of  the  processes  of 
the  healthy  body  we  can  hardly  hope  to  diagnose  and  treat  its 
derangements  when  diseased;  and  our  physicians  are  in  many 
cases  competent  physiologists.  But  it  is  equally  important  to 
understand  the  method  in  which  the  diseased  body  combats  and 
cures  an  infection  ;  and,  although  our  knowledge  of  this  is  as  yet 
imperfect,  it  is  increasing  day  by  day,  and  results  of  the  greatest 
interest  to  the  practising  physician  have  already  been  obtained. 
And  I,  for  one,  think  that  an  intelligent  appreciation  of  what  is 
actually  taking  place  in  the  body,  of  the  conservative  and  adverse 
forces,  and  of  the  conditions  necessary  for  cure,  will  always  be  of 
value  to  the  therapist,  although  it  may  not  give  any  definite 
information  as  to  what  drug  is  to  be  prescribed. 

Let  us  revert  to  the  subject  of  NATURAL  IMMUNITY.  We  may 
define  it  roughly  as  the  immunity  possessed  by  a  certain  individual 
in  virtue  of  its  belonging  to  a  given  animal  species  ;  it  is  inherent 
to  a  greater  or  less  extent  in  all  members  of  that  species,  and  is  not 
dependent  on  any  event  taking  place  during  the  life  of  the  animal 
in  question.  In  most  cases  it  is  present  at  birth,  though  this  is 
not  absolutely  essential. 

Examples  are  numerous.  The  lower  animals  are  immune  to 
the  gonococcus,  and,  with  few  exceptions  (the  higher  apes),  to 
syphilis  also.  On  the  other  hand,  most  of  the  diseases  of  the 
lower  animals  do  not  affect  man — fowl  cholera,  canine  distemper, 
and  rinderpest  are  a  few  of  many  examples.  In  some  cases  all 
animals,  with  a  few  exceptions,  are  immune :  this  is  the  case  with 
the  venereal  diseases,  and  in  some  of  the  protozoal  infections  of 


8  NATURAL   IMMUNITY 

the  lower  animals.  In  others  different  types  of  the  infecting 
organism  occur,  and  a  given  species  is  susceptible  to  one,  immune 
to  others  ;  for  example,  there  are  three,  and  perhaps  more,  varieties 
of  tubercle  bacillus,  which  resemble  one  another  in  many  points, 
and  which  attack  respectively  man,  cattle,  and  birds,  and  each 
animal  species  is  more  or  less  immune  to  bacilli  from  animals  far 
removed  in  the  scale. 

In  general  terms,  the  immunity  or  susceptibility  of  different 
animals  depends  to  some  extent  on  their  zoological  affinities. 
Thus  man  is  pre-eminently  susceptible  to  the  Spirochata  pallida, 
the  anthropoid  apes  less  so,  but  still  not  immune,  and  the  lower 
animals  entirely  refractory.  Rinderpest  affects  cattle,  sheep, 
goats,  and  other  ruminants,  and  South  African  horse-sickness 
horses,  asses,  and  mules.  But  to  this  rule  there  are  numerous 
exceptions  :  thus,  almost  all  warm-blooded  animals  are  susceptible 
to  anthrax,  but  the  Algerian  sheep  and  white  rat  are  relatively 
immune,  the  wild  rat  being  susceptible.  And  of  the  domestic 
animals  we  find  cattle  to  be  highly  susceptible  to  tubercle,  whereas 
goats,  though  closely  allied  zoologically,  are  almost  immune. 

Natural  immunity  does  not  exist  to  an  equal  degree  in  all 
individuals  of  a  species.  This  is  well  seen  in  man  during  an 
epidemic,  where,  of  a  certain  number  of  persons  who  are  exposed 
to  an  infection  (and,  as  far  as  we  know,  receive  the  same  dose  of 
the  materies  morbi),  some  escape  the  disease  altogether,  some  have 
a  slight,  and  others  a  severe,  attack,  whilst  yet  others  die  rapidly. 
Sex  has  some  influence  here,  but  it  is  usually  difficult  to  trace, 
since  the  males  and  females  of  a  community  are  in  most  cases 
exposed  to  an  infection  in  varying  degree. 

Age  is  of  more  importance,  and,  in  quite  general  terms,  we  may 
say  that  the  younger  the  infant  the  less  its  immunity.  Certain 
diseases,  such  as  measles,  scarlet  fever,  and  whooping-cough,  are 
rarely  seen  except  in  infants,  and  this  is  not  altogether  due  to 
acquired  immunity  preventing  a  second  attack  in  later  life. 
Epidemic  diarrhoea  due  to  bacilli  of  the  dysentery  group  is 
rarely  seen — in  this  country,  at  least — except  in  the  early  years  of 
life,  and  the  same  is  true  of  cerebro-spinal  meningitis  and  some 
other  diseases.  It  is  also  interesting  to  notice  that  the  variation 
in  immunity  may  take  a  qualitative  rather  than  a  quantitative 
form.  The  best  example  is  in  the  case  of  the  pneumococcus. 
This  organism  is  the  chief  cause  of  suppurative  processes  of 
whatever  region  in  infants,  whereas  in  adults  it  is  (except  in 


INTRODUCTORY   AND   GENERAL  9 

certain  regions)  a  decidedly  rare  cause  of  abscesses  and  other 
pyogenic  processes.  It  is  evident  that  the  form  of  immunity 
which  prevents  the  pneumococcus  from  gaining  access  to  the 
tissues  and  giving  rise  to  abscess  formation  is  in  abeyance  in  the 
young  and  well  developed  in  the  adult ;  yet  the  two  are  more 
nearly  equal  in  their  resistance  to  this  organism  in  its  role  of  a 
producer  of  pneumonia.  There  are  also  very  marked  differences 
in  regard  to  local  immunity  in  the  two  ages,  but  of  those  we  shall 
speak  subsequently. 

Natural  immunity  must  not  be  regarded  as  a  fixed  and  definite 
quantity,  since  all  individuals  vary  enormously  in  their  resisting 
powers  against  various  diseases  at  different  times  and  under 
different  conditions.  The  factors  which  tend  to  break  down  the 
immunity  against  any  or  all  infections  may  be  referred  to  as  the 
banal  causes  of  the  diseases  in  question.  They  are  not  in  them- 
selves sufficient  to  lead  to  these  diseases,  but  when  they  come 
into  action  and  an  infecting  agent  is  present  the  disease  will  arise. 
Hence  they  are  often  referred  to  as  predisposing  causes  of  disease, 
and  to  the  lay  public  they  are  the  actual  causes,  since  they  are 
usually  open  and  obvious,  and  the  real  infecting  agent  is,  of 
course,  unknown  They  are  of  the  utmost  importance  in  pre- 
ventive medicine,  and  wherever  the  probability  of  an  infection  is 
apprehended,  a  study  of  the  patient's  surroundings  and  habits 
may  often  lead  to  the  giving  of  advice  by  which  these  banal 
causes  of  infection  may  be  avoided  and  the  disease  warded  off. 
In  general  these  predisposing  causes  are  a  study  for  the  physician 
rather  than  for  the  pathologist,  and  in  some  cases  we  are  quite  in 
the  dark  as  to  the  method  in  which  they  act.  Their  study  cannot 
be  conveniently  undertaken  here  before  the  mechanisms  and  pro- 
cesses of  immunity  have  been  described,  but  it  will  be  useful  to 
enumerate  some  of  the  more  important. 

Of  these  cold  and  wet,  especially  in  combination,  are  unquestion- 
ably the  most  important.  The  exact  way  in  which  they  act  is 
not  definitely  known,  but  there  are  materials  for  a  number  of 
suggestions.  Thus,  as  we  shall  have  abundant  opportunity  of 
seeing,  immunity  is  to  a  very  large  extent  a  function  of  the  leuco- 
cytes, to  which  are  specialized  cells  to  which  the  defence  of  the 
body  is  entrusted.  Now  the  functions  (movement  and  phagocy- 
tosis) which  can  be  easily  investigated  are  found  to  be  dependent 
in  a  very  high  degree  on  temperature,  acting  best  at  the  tempera- 
ture of  the  body,  or  slightly  above  ;  and  it  is  highly  probable  that 


10  COLD,    WET,    AND    FATIGUE 

the  more  subtle  functions  of  the  leucocytes  may  be  similarly 
depressed  by  a  low  temperature.  The  exposure  of  the  skin  to 
cold,  especially  if  the  animal  heat  be  abstracted  more  quickly 
by  evaporation  of  moisture  on  the  surface,  will  lead  to  a  cooling 
of  the  blood  which  circulates  through  it,  and  hence  to  a  slight, 
though  appreciable,  cooling  of  the  whole  blood.  This,  it  is  true, 
is  soon  compensated  for,  and  no  great  amount  of  cooling  of  the 
whole  body  occurs;  but  even  so,  it  is  quite  possible  that  the 
periodical  chilling  of  the  leucocytes  during  their  repeated  passages 
through  the  cold  skin  may  be  sufficient  to  diminish  greatly  their 
functional  activity,  and  to  lower  the  resistance  to  a  point  at  which 
infection  can  occur,  and  when  once  pathogenic  bacteria  have 
gained  a  foothold,  the  resistance  will  for  a  time  tend  to  decrease. 
There  is  also  some  evidence  going  to  show  that  exposure  to  cold 
may  lessen  the  production  of  the  defensive  substances  which  occur 
in  the  blood  (alexin,  antibodies,  etc.),  though  this  is  not  fully 
proved.  It  is  worthy  of  note  that  the  loss  of  immunity  due  to  the 
action  of  cold  and  wet  on  one  part  of  the  body  (such  as  the  feet) 
is  a  general  one,  and  may  result  in  a  nasal  catarrh,  an  attack  of 
pneumonia,  acute  rheumatism,  etc.,  according  to  the  nature  of  the 
infection  at  hand.  It  is  not  necessarily  a  local  infection  of  the 
chilled  region.  This  is  very  well  shown  experimentally.  Fowls 
are  immune  to  anthrax,  but  are  rendered  susceptible  if  they  are 
kept  for  some  time  standing  in  cold  water ;  and  this  acquired 
susceptibility  is  then  a  general  one,  and  not  merely  of  the  feet. 

Cold  and  wet,  as  is  well  known,  have  less  action  when  accom- 
panied by  energetic  muscular  exercise,  so  long  as  this  does  not 
reach  the  extent  of  undue  fatigue.  This  is  not  because  less  heat 
is  lost  during  exercise.  The  reverse  is  the  case.  The  suggested 
explanation  is  that  the  muscular  metabolism  leads  to  an  increased 
production  of  heat,  and  at  the  same  time  the  cutaneous  capillaries 
are  dilated  and  the  heart  accelerated,  or  that  the  circulation  of 
blood  through  the  skin  occurs  quickly ;  further,  the  internal 
temperature  of  the  body  may  actually  be  raised  several  degrees. 
The  result  is  that  the  temperature  of  any  given  leucocyte  never 
falls  much  below  normal,  if  at  all,  since  it  comes  from  the  internal 
regions  where  the  temperature  is  raised,  passes  rapidly  through 
the  skin,  and  returns  again  to  the  interior  of  the  body. 

The  effect  of  fatigue,  either  alone  or  in  conjunction  with  cold  and 
wet,  is  also  well  known,  and  is  one  reason  for  the  excessive  mor- 
tality from  disease  of  armies  in  the  field.  It  is  less  explicable, 


INTRODUCTORY   AND   GENERAL  II 

but  may  probably  be  connected  in  some  way  with  the  presence  in 
the  blood  of  katabolic  products  of  muscular  activity,  which  have 
an  injurious  action  on  the  cells  of  the  tissues  in  general  and  on  the 
leucocytes  in  particular.  Further,  the  metabolic  products  formed 
during  the  action  of  the  muscles  are  acid  in  reaction,  and  it  is 
found  that  some  at  least  of  the  protective  substances  which  occur 
in  the  blood  (alexins  and  opsonins)  act  best  in  an  alkaline  medium. 
This  diminution  of  immunity  after  muscular  fatigue  is  manifested 
in  animals  as  well  as  in  man.  White  rats  which  have  been  made 
to  work  in  a  revolving  cage  are  more  susceptible  to  anthrax  than 
normal  white  rats,  the  pre-existing  immunity  being  broken  down. 

Insufficient  or  unsuitable  food  is  a  factor  of  importance,  especially, 
perhaps,  in  the  aetiology  of  tuberculosis.  It  is,  however,  rarely 
seen  alone — in  this  country,  at  any  rate — and  in  the  poorer  classes 
its  effects  are  usually  complicated  by  insufficient  clothing,  un- 
cleanly habits,  and  by  insufficient  ventilation  of  their  houses.  For 
this  reason  we  may  perhaps  be  led  to  exaggerate  its  importance ; 
and  whilst  it  is,  of  course,  true  that  semi-starvation,  in  common 
with  other  weakening  influences,  does  pave  the  way  for  infective 
processes,  we  do  not  find  that  a  supply  of  food  restricted  enough 
to  cause  a  marked  reduction  of  the  bodily  strength  and  some 
degree  of  anaemia  is  necessarily  associated  with  any  infective 
disease,  though  the  patient  may  live  under  conditions  in  which 
infective  material  is  present  in  abundance.  This  is  well  seen  in 
fasting  men,  in  hysterical  anorexia,  and  in  patients  with  imperme- 
able cesophageal  strictures.  The  blood,  it  may  be  pointed  out,  is 
not  one  of  the  tissues  that  suffers  first  in  starvation,  and  its  im- 
portance to  the  body  in  many  ways  is  so  great  that  it  is  kept  in 
good  functional  activity  whilst  other  regions  waste  quickly. 

It  is  probable  that  insufficient  food  lowers  the  resistance  of  the 
body  in  certain  directions  rather  than  in  others.  In  the  East 
plague  follows  famine  with  some  regularity,  but  there  is  little  or 
no  connection  between  famine  and  cholera.  But  in  these  latitudes 
at  the  present  time  the  disease  most  commonly  due  to  bad  or  in- 
sufficient food  is  tuberculosis.  Formerly  it  was  relapsing  fever,  or, 
as  it  was  sometimes  called,  famine  fever,  a  disease  which  is  now 
almost  extinct  as  a  result  of  the  general  cheapening  of  foodstuffs. 

It  is  worthy  of  note  that  the  number  of  leucocytes  per  cubic 
centimetre  diminishes  in  starvation,  and  is  generally  lower  in  the 
badly-nourished  than  in  the  well-fed  ;  and  these  cells,  as  we  shall 
see,  are  pre-eminently  concerned  in  immunity,  and  this  in  a  great 


12  EFFECTS    OF    A   VITIATED    ATMOSPHERE 

many  ways.  It  was  recognized  long  ago  that  post-mortem  wounds 
are  much  more  dangerous  when  received  whilst  fasting  than  during 
the  process  of  digestion,  and  it  is  possible  that  this  may  be  due  to 
some  extent  to  the  increased  number  of  leucocytes  which  occur  in 
the  blood  during  the  process. 

Exposure  to  a  vitiated  atmosphere,  if  of  long  duration,  is  a  most 
potent  cause  of  the  breaking  down  of  immunity,  and  when  con- 
sidered on  a  large  scale,  and  in  view  of  its  effect  on  the  general 
death  and  disease  rate,  is  probably  of  greater  importance  than  all 
other  causes  combined.  It  is  especially  important  in  connection 
with  tuberculosis,  and  nothing  is  more  striking  than  to  notice  its 
effect  on  the  peasantry  of  some  regions,  in  which,  in  spite  of 
exposure  to  abundant  fresh  air  during  the  daytime,  and  a  supply 
of  food  which  certainly  does  not  fall  below  the  physiological 
minimum,  and  is  usually  more  abundant,  phthisis  and  other 
tuberculous  diseases  are  rife.  These  affections  are  in  general 
common  in  cold  and  windy  climates,  and  less  prevalent  in  warmer 
countries,  and  there  is  little  doubt  that  the  main  reason  for  this  is 
the  habit  which  dwellers  in  cold  countries  frequently  contract  of 
hermetically  sealing  all  entrances  to  their  rooms  to  keep  out  the 
cold.  But  this  is  frequently  seen  in  warmer  regions,  and  even 
throughout  the  South  of  England  there  is  an  almost  universal 
opinion  amongst  the  lower  classes  that  night  air  is  injurious. 
This  is  probably  a  survival  from  the  time  when  malaria  was 
indigenous  in  this  country. 

Apart  from  tubercle,  the  effect  of  bad  air  is  especially  mani- 
fested in  the  causation  of  diseases  of  the  lungs,  nose,  throat,  etc., 
and  its  effect  is  probably  partly  general  and  partly  local.  The 
effect  of  irritating  vapours  is,  of  course,  local.  Thus  exposure  to 
nitrous  fumes  is  often  followed  by  the  rapid  development  of 
pneumonia,  and  this  is,  or  may  be,  due  to  the  pneumococcus, 
which  is  able  to  invade  the  injured  lung. 

We  do  not  know  the  mechanism  by  which  ordinary  vitiated  air 
acts  on  the  general  immunity. 

Prolonged  anesthesia  is  probably  a  cause  of  considerable 
importance,  though  one  not  easy  to  estimate.  The  prevalence  of 
ether-pneumonia  is  not  yet  ascertained,  and  has  been  hotly 
debated.  It  falls,  of  course,  into  the  same  category  as  the 
pneumonia  due  to  irritating  vapours,  as  described  above.  Apart 
from  this,  however,  there  is  reason  to  believe  that  prolonged 
anaesthesia  has  some  effect  in  lowering  the  general  resisting 


INTRODUCTORY   AND    GENERAL  13 

power  of  the  body  to  the  common  pyogenic  bacteria,  and  that  the 
mere  length  of  an  operation  should  be  an  indication  for  the  most 
scrupulous  care  in  antiseptic  precautions.  It  is  perhaps  con- 
ceivable that  the  anaesthetic  drug  present  in  the  blood  may  be 
sufficient  to  paralyze  the  leucocytes  for  a  sufficient  time  to  allow 
bacteria  to  gain  a  foothold  in  the  body. 

Certain  drugs,  of  which  the  most  important  is  alcohol,  have  an 
important  action  in  this  respect.  The  liability  of  alcoholic 
subjects  to  pneumonia  and  some  other  infective  diseases  is  well 
known,  and  in  them  the  prognosis  is  more  than  usually  unfavour- 
able. We  have  but  little  knowledge  of  the  action  of  alcohol  in 
this  respect.  It  may  be  that  it  acts  as  a  direct  inhibitant  of  the 
activity  of  the  leucocytes,  and  it  is  known  to  destroy  certain 
delicate  defensive  substances  (alexins  and  opsonins)  which  play 
some  part  in  the  defence  of  the  body  against  microbic  invasion, 
but  it  is  not  known  whether  these  effects  are  actually  manifested 
in  the  circulating  blood.  It  is  also  possible  that  alcohol  tends  to 
inhibit  the  formation  of  these  defensive  substances. 

Alcohol  tends  to  lower  the  temperature  of  the  body  by  increas- 
ing the  amount  of  heat  lost.  It  dilates  the  superficial  vessels  and 
accelerates  the  heart's'  action  in  a  way  somewhat  similar  to 
muscular  exercise,  but  does  not,  like  it,  raise  the  temperature  of 
the  interior  of  the  body.  Hence  the  effect  of  alcohol  in  conjunc- 
tion with  cold  and  wet  is  to  increase  their  ill-effects.  More  blood 
is  forced  through  the  chilled  skin  and  more  heat  is  lost.  The 
injurious  effect  of  alcohol  during  exposure  to  cold  is  well  known. 
The  results,  however,  are  different  when  alcohol  is  taken  after 
exposure,  and  when  the  sufferer  has  reached  warmth  and  shelter. 
There  the  increased  flow  in  the  cutaneous  capillaries  leads  to  a 
warming  of  the  skin  and  consequent  cessation  of  the  chilling  of 
the  blood,  although  the  loss  of  heat  may  go  on. 

Diseases — the  most  important  of  which  are  Bright's  disease  and 
diabetes — lead  to  a  general  lowering  of  the  level  of  immunity,  and 
a  consequent  predisposition  to  other  diseases.  We  have  no 
knowledge  of  the  way  in  which  they  act. 

There  are  many  causes  which  act  locally,  and  cause  a  local 
lowering  of  the  resistance.  Some  of  these  have  been  hinted  at 
above,  but  their  consideration  will  be  deferred  for  the  present. 

In  considering  the  nature,  severity,  and  prognosis  of  any  disease, 
two  factors  have  to  be  recognized  :  (i)  the  immunity  of  the  patient, 


14  VIRULENCE   OF   BACTERIA 

and  (2)  the  virulence  of  the  infecting  bacterium.  A  third — the 
number  of  bacteria  which  gain  access — is  also  of  importance, 
especially  under  experimental  conditions,  for  it  is  found  that, 
within  limits,  lack  of  virulence  can  be  compensated  for  by  an 
increase  in  the  dose  given.  It  is,  however,  one  which  we  can 
rarely  estimate  in  natural  disease ;  besides  which  the  growth  of 
bacteria  is  so  rapid  that,  if  not  checked  by  the  resisting  power  of 
the  body,  a  single  organism  would  multiply  in  a  very  few  hours 
to  an  enormous  extent,  and  render  it  a  matter  of  but  little  impor- 
tance whether  one  or  a  hundred  bacteria  had  gained  access  at 
first.  The  number  of  bacteria  is  probably  of  more  importance  in 
connection  with  the  occurrence  or  non- occurrence  of  infection, 
rather  of  the  severity  of  the  disease  when  once  infection  has 
occurred.  Thus  we  find  in  epidemics  of  typhoid  fever  due  to 
water  or  milk  that  the  disease  is  most  prevalent  in  those  who 
take  a  large  amount  of  the  infective  material,  but  it  is  not  neces- 
sarily more  severe  in  them  than  in  the  patients  who  have  appa- 
rently become  infected  with  a  small  dose.  This  is,  however,  not 
the  case  with  artificial  infection  of  animals,  for  there  the  severity 
of  the  disease  (in  animals  as  similar  as  possible  in  age,  weight, 
etc.)  is  fairly  proportional  to  the  dose  given.  But  the  conditions 
are  somewhat  different  in  the  two  cases,  and  in  the  artificial  injec- 
tion of  animals  we  eliminate  altogether  the  steps  by  which,  e.g., 
the  typhoid  bacillus  passes  the  natural  barriers,  and  gains  access 
to  the  tissues. 

The  question  of  virulence  is  of  much  greater  importance,  and 
is  one  which  must  be  more  fully  discussed  subsequently,  after  we 
have  seen  the  methods  in  which  the  host  immunizes  itself  against 
the  bacterium.  Some  general  points  must  be  mentioned  here. 

Cultures  of  the  same  organism,  identical  in  all  respects  in 
morphological,  cultural,  and  chemical  characters,  may  differ 
enormously  in  this  respect :  thus  a  culture  of  streptococci  may  be 
entirely  devoid  of  virulence  to  rabbits,  or  may  be  so  potent  that  a 
minimal  dose,  containing  probably  but  a  single  coccus  or  short 
chain,  may  be  inevitably  fatal.  Similar  facts  hold  for  pneumo- 
cocci.  According  to  Eyre,  a  virulent  culture  may  kill  when  20  to 
200  cocci  are  injected,  whereas  an  avirulent  one  may  fail  to  do 
so  in  massive  doses.  In  most  organisms  there  is,  perhaps,  not 
such  a  marked  difference,  but  all  pathogenic  bacteria  vary  greatly 
in  this  respect,  and  cultures  from  different  sources  show  marked 
variations  in  pathogenicity. 


INTRODUCTORY  AND   GENERAL  15 

Further,  the  same  culture  can  be  made  to  undergo  variation, 
its  virulence  being  either  exalted  or  diminished,  and  this  is  a 
subject  of  the  utmost  importance.  An  increase  in  virulence  is  the 
more  difficult  to  secure,  and  can  practically  only  be  procured  by 
passage  through  animals,  or  by  other  closely  allied  process. 

Passage  is  carried  out  thus  :  the  avirulent  culture  is  made  to 
infect  animals  either  by  the  administration  of  massive  doses,  or 
by  the  simultaneous  injection  of  some  substance  which  lowers  the 
local  or  general  resistance  (lactic  acid,  alcohol,  the  toxins  of 
B.  prodigiosus,  etc.).  In  any  case,  the  organism  is  made  to  cause 
an  infection  which  may  or  may  not  be  allowed  to  progress  to  a 
fatal  issue.  From  the  animal  thus  infected  a  second  culture  is 
made,  and  the  material  used  to  inoculate  a  second  animal,  and 
the  organism  will  be  found  to  have  undergone  a  noticeable  access 
of  virulence.  The  process  is  repeated  as  often  as  is  necessary, 
and  ultimately  the  virulence  of  the  culture  will  be  brought  to 
its  highest  possible  pitch.  The  simplest  method,  where  available, 
is  to  give  the  injections  into  the  peritoneum,  and  to  make  the 
cultures  by  withdrawing  some  of  the  peritoneal  fluid  in  a  sterile 
pipette,  and  incubating  it  as  it  is,  or  after  the  addition  of  broth. 

This  method  was  introduced  by  Pasteur,  and  is  of  especial 
value  in  preparing  the  vaccine  used  against  rabies.  The  organism 
of  this  disease  is  unknown,  but  the  virus  occurs  in  the  brain,  and 
emulsions  of  this  substance  are  used  for  inoculation.  It  is  found 
that  the  virus  occurring  naturally  in  rabid  dogs  (the  "  virus  of 
the  streets ")  is  comparatively  avirulent  to  rabbits.  This  is 
shown  by  the  long  incubation  period — fifteen  to  eighteen  days 
after  intracerebral  injection.  After  about  fifty  passages  through 
rabbits,  the  virus  becomes  so  exalted  that  the  incubation  period 
is  shortened  to  six  days,  and  the  process  cannot  be  carried  further. 
This  virus  is  called  the  "  fixed  virus,"  and  its  potency  is  main- 
tained unaltered,  no  matter  how  many  more  passages  are  made. 

Passage  does  not  necessarily  raise  the  virulence  of  the  culture 
to  all  animals  ;  it  may  do  so  only  for  the  species  used  for  the  pro- 
cess, the  action  on  other  species  remaining  unaltered  or  even 
falling.  Nor  is  passage  necessarily  followed  by  an  increased 
degree  of  virulence — the  virus  of  rabies  diminishes  in  this  respect 
when  passed  through  apes. 

Phenomena  suggesting  a  process  akin  to  passage  occur  under 
natural  conditions.  Pneumococci  are  frequently  found  in  the 
mouths  of  healthy  persons,  and  are,  as  a  rule,  of  feeble  virulence, 


l6  INCREASED   VIRULENCE 

whilst  those  which  are  isolated  from  the  lungs  in  pneumonia,  or 
from  pneumococcic  lesions  in  general,  are  usually  far  more  virulent. 
Other  explanations  are  possible,  but  it  seems  likely  that  the 
sequence  of  events  is  as  follows  :  The  avirulent  pneumococci  gain 
access  to  the  body  owing  to  a  temporary  loss  of  immunity,  due  to 
one  or  other  of  the  causes  enumerated  above,  and  then  these  are 
transmitted  to  a  process  in  all  respects  like  passage,  the  result 
being  that  they  undergo  a  gradual  increase  in  virulence.  The 
struggle  of  the  conservative  forces  will  then  be  increasingly  difficult, 
and  the  patient  may  succumb  to  an  infection  with  an  organism 
which  was  at  first  but  slightly  virulent.  This  adaptation  of  an 
organism  to  its  environment  during  the  course  of  a  disease  may 
probably  be  found  in  the  future  to  be  of  great  importance,  as  indi- 
cating a  necessity  for  successive  changes  in  the  vaccine  or  serum 
used  in  the  treatment  of  a  chronic  infection. 

An  example  worthy  of  notice  has  recently  been  given  by 
Ehrlich.  It  is  not  exactly  on  the  same  lines,  since  it  deals  with 
an  alteration  in  the  body  of  the  power  possessed  by  the  parasite  of 
resisting  chemical  agents  of  relatively  simple  composition,  rather 
than  in  the  power  of  resisting  the  natural  forces  of  the  body,  an 
increase  in  which  constitutes  an  increase  in  virulence.  Ehrlich 
investigated  the  preventive  and  curative  action  of  atoxyl  and  of 
various  aniline  dye-stuffs,  such  as  fuchsin  and  trypanroth,  on  mice 
infected  with  trypanosomiasis.  He  found  in  a  certain  number  of 
cases  a  cure  might  be  obtained — e.g.,  by  feeding  infected  mice  with 
fuchsin  or  by  the  injection  of  atoxyl — and  that  when  this  occurred 
the  trypanosomes  were  not  entirely  destroyed,  but  remained  latent 
in  the  body.  This  is  a  phenomenon  of  fairly  frequent  occurrence, 
and  is  called  by  Ehrlich,  "  immunitas  non  sterilisans."  After  a 
time  a  relapse  occurred,  and  was  cured  by  a  fresh  dose  of  the  drug, 
but  after  several  of  these  recurrences  this  beneficial  effect  ceased. 
It  was  then  found  that  the  trypanosomes  had  been  immunized  or 
acclimatized  to  the  agent  in  question — say,  to  fuchsin — and  pos- 
sessed the  power  of  infecting  mice  previously  treated  with  fuchsin 
and  immune  to  ordinary  trypanosomes ;  but  the  organism  had 
not  altered  in  its  susceptibility  to  other  dye-stuffs  or  to  atoxyl, 
and  mice  infected  with  it  could  be  cured  by  these  agents,  and  not 
by  fuchsin.  Further,  it  was  found  possible  to  create  a  race  of 
trypanosomes  resistant  to  two  or  more  of  these  agents,  and  these 
acquired  characters  were  made  permanent  after  several  passages. 
If  we  substitute  for  the  drugs  used  by  Ehrlich  the  substances 


INTRODUCTORY   AND    GENERAL  17 

which  are  developed  in  the  body  as  defensive  agents  during  an 
attack  of  disease,  and  imagine  the  same  process  to  go  on,  we 
shall  have  an  exact  reproduction  of  the  rise  in  virulence  occurring 
during  an  attack  of  disease.  If  we  defended  ourselves  against 
trypanosomes  by  the  development  of  fuchsin,  Ehrlich's  fuchsin- 
resistant  race  would  be  extremely  virulent  for  us. 

The  development  of  epidemics  of  diseases  is  probably  due  in 
some  cases  to  a  spontaneous  rise  in  virulence  of  the  infecting  agent, 
but  we  have  no  knowledge  of  the  causes  by  which  this  is  produced. 

The  second  method  of  increasing  the  virulence  of  a  culture  is 
less  general,  and  of  greater  theoretical  than  practical  interest.  It 
consists  in  the  cultivation  of  the  organism  for  several  generations 
in  the  blood-serum  of  an  animal  which  has  been  immuned  to  the 
bacterium  in  question.  It  was  discovered  by  Walker  in  the  case 
of  B.  typhosus,  and  is  found  in  the  case  of  some  other  organisms. 
It  is  referred  to  subsequently,  and  we  need  only  say  here  that  it  is 
allied  to  passage ;  the  organism  is  immunized  to  the  fluids  of  the 
resistant  animal  in  vitro  instead  of  in  vivo.  And  the  virulence  of  a 
culture  is  in  general  best  sustained  by  a  close  approximation  to 
the  conditions  of  the  body.  Thus  it  is  more  rapidly  lost  at  the 
temperature  of  the  room  than  at  that  of  the  body,  and  the  most 
suitable  culture  medium  is  usually  one  containing  body  fluids 
unaltered  by  heat.  Thus  Marmorek  cultivates  his  virulent  strepto- 
cocci in  broth  to  which  one-third  of  its  volume  of  ascitic  fluid  has 
been  added.  In  the  case  of  diphtheria  bacilli  the  virulence  (as 
estimated  by  its  power  of  forming  toxin)  is  best  maintained  by 
daily  transplantations  into  broth  previously  raised  to  the  body 
temperature,  and  when  treated  in  this  way  shows  little  or  no 
change  for  years. 

Diminution  in  virulence  occurs,  as  a  rule,  when  the  organism  is 
submitted  to  conditions  quite  unlike  those  of  the  animal  body,  and 
is  usually  the  more  rapid  the  greater  the  divergence.  At  the  same 
time,  the  growth  under  these  conditions  gradually  becomes  (in 
most  cases)  more  abundant.  The  organism  gradually  adapts  itself 
to  a  saprophytic  habitat,  losing  in  so  doing  its  distinctive  chemical 
properties  which  made  it  virulent  as  a  parasite.  Old  laboratory 
cultures  of  bacteria  which  have  been  grown  on  artificial  media  for 
many  generations  are  usually  almost  devoid  of  virulence,  though 
here  there  are  great  variations,  some  species  becoming  inert  far 
quicker  than  others. 

The  subject  is  important,  since  cultures  of  diminished  ("  miti- 

2 


l8  DIMINUTION    IN   VIRULENCE 

gated  ")  virulence  are  frequently  employed  as  vaccines  in  the  pro- 
duction of  artificial  immunity.  The  following  are  some  of  the 
chief  methods  employed : 

1.  By  prolonged  culture  in  artificial  media,  as  described  above. 
This   method   was  introduced  by  Pasteur  in   the  case  of  fowl 
cholera.     The  loss  of  virulence  is  a  progressive  one,  and  cultures 
ten  months  old  are  devoid  of  virulence. 

2.  By  cultivating  the  organism  at  a  temperature  above  the 
optimum  for  saprophytic  growth.     This  was  also  introduced  by 
Pasteur,  and  is  used  in  preparing  the  vaccines  to  anthrax.     The 
organism   is   cultivated   at   a  temperature  of  42-5°  C.,  and  all 
virulence  is  destroyed  in  about  six  weeks,  though  the  cultures 
retain  their  power  of  growth  unaltered.  The  first  vaccine  is  prepared 
by  allowing  growth  to  continue  at  this  temperature  for  twenty- 
four  days.     In  appearance  the  bacilli  are  unaltered,  but  they  have 
lost  the  power  of   killing  rabbits  and  guinea-pigs,  though  they 
are  still  fatal  to  mice.     The  second  vaccine  is  cultivated  at  a  high 
temperature   for   a   fortnight   only;    it  is  virulent  to   mice   and 
guinea-pigs,  but  not  to  rabbits. 

The  process  may  also  be  carried  out  by  a  short  exposure  to  a 
higher  temperature.  Chauveau's  vaccine  consists  of  blood  con- 
taining anthrax  bacilli,  heated  to  a  temperature  of  50°  to  55°  C.  for 
ten  or  fifteen  minutes.  The  bacilli  remain  alive,  but  are  mitigated 
in  virulence. 

3.  In  some  cases  the  addition  of  various  chemical  antiseptics  in 
minute  amounts  to  the  culture  medium  has  a  similar  effect.    This 
is  the  case  with  anthrax  also.     Addition  of  chemical  substances  is 
also  made  with  the  idea  of  destroying  toxins,  but  this  is  a  different 
phenomenon. 

4.  The  virulence  may  be  destroyed  by  drying.     This  method 
was  introduced  by  Pasteur  for  the  preparation  of  a  vaccine  against 
rabies.     We  have  already  described  the  method   by  which  he 
obtained  the  fixed  virus  and  its  action  on  rabbits.    He  found  that 
by  suspending  the  spinal  cords  of  rabbits  dead  of  this  fixed  virus 
over  caustic  potash  at  a  temperature  of  23°  C.  the  virulence  was 
entirely  removed  in  fifteen  days.      Drying   for   a   shorter  time 
diminished  the  virulence,  but  did  not  remove  it  entirely.1 

1  The  more  modern  idea  is  that  the  process  of  drying  kills  off  a  large 
number  of  the  pathogenic  organisms,  and  that  the  use  of  the  dried  cord  is 
merely  another  method  of  giving  very  minute  doses  of  virus  of  normal 
strength. 


INTRODUCTORY   AND    GENERAL  IQ 

5.  In  some  cases  (as  has  been  noted  above)  passage  through 
animals  diminishes  the  virulence.  In  some  cases  this  can  be 
exalted  by  passage  through  a  series  of  animals  of  one  species  and 
diminished  by  the  use  of  another.  Pasteur  showed  this  to  be  the 
case  in  swine  erysipelas,  the  potency  of  which  (as  tested  on  pigs) 
is  increased  by  passage  through  pigeons  and  decreased  by  passage 
through  rabbits.  Cultures  thus  attenuated  are  used  as  vaccines. 

The  term  ACQUIRED  IMMUNITY  is  one  that  is  used  to  denote  an 
increased  resistance  to  an  organism  dependent  on  some  modifica- 
tion in  the  animal's  constitution  due  to  some  definite  process  to 
which  it  is  subjected,  but  not  including  the  modifications  due  to 
improvements  in  the  general  health  due  to  betterment  of  the 
environment.  For  example,  a  person  living  in  insanitary  sur- 
roundings will  undoubtedly  acquire  a  higher  degree  of  resistance 
to  the  tubercle  bacillus  on  being  moved  to  more  healthy  ones,  but 
we  do  not  speak  of  that  as  acquired  immunity.  The  distinction 
is  this:  The  elevation  of  the  natural  resisting  powers  due  to 
improvement  in  the  general  vitality  is  a  more  or  less  general  one, 
and  affects  the  immunity  to  most  or  all  bacteria  almost  equally ; 
whereas  in  acquired  immunity  in  the  narrower  sense,  to  which 
the  use  of  the  term  is  restricted  by  pathologists,  the  alteration  is 
in  the  powers  of  resistance  to  one  bacterium  only.  For  example, 
a  debilitated  person  removed  to  a  more  healthy  environment, 
given  better  food,  tonics,  etc.,  would  become  more  resistant  to 
the  attacks  of  smallpox,  and  to  other  diseases  as  well ;  we  should 
speak  of  that  as  an  augmentation  of  the  natural  immunity.  But 
after  an  attack  of  smallpox,  or  after  vaccination,  his  immunity 
to  smallpox  is  enormously  increased,  whereas  his  resistance  to 
other  organisms  is  unaltered  ;  this  is  acquired  immunity. 

This  is  expressed  by  the  use  of  the  word  specific,  embodying  an 
idea  difficult  to  define,  but  implying  a  direct  relationship  of  cause 
and  effect,  and,  moreover,  that  a  certain  effect  is  only  produced 
by  a  certain  definite  cause.  Thus  the  toxin  of  diphtheria  is 
specific  for  the  diphtheria  bacillus  in  the  sense  that  it  is  produced 
by  that  organism,  and  by  no  other  ;  diphtheria  antitoxin  is  specific 
for  diphtheria  toxin,  since  it  is  produced  only  as  a  result  of  the 
injection  of  that  substance ;  the  reaction  caused  by  the  injection 
of  tuberculin  into  a  tuberculous  animal  is  specific,  etc. 

In  most  instances  there  is  another  difference  between  a  rise  in 
natural  immunity  and  the  development  of  acquired  immunity,  in 
that  the  latter  is  much  stronger.  Thus,  the  power  of  resistance 


20  ACQUIRED   IMMUNITY 

to  smallpox  of  a  perfectly  healthy  person  is  probably  not  great, 
whereas  that  produced  by  an  attack  of  the  disease  or  by  vacci- 
nation is  for  a  time  almost  absolute.  Yet  all  degrees  of  acquired 
immunity  exist,  from  the  very  slight  amount  which  is  developed 
during  an  attack  of  pneumonia,  and  which  is  probably  only  just 
sufficient  to  cut  short  the  disease,  to  the  enormous  degree  that 
can  be  obtained  in  animals  hyperimmunized  to  diphtheria  or 
tetanus  toxin  or  hypervaccinated  to  B.  typhosus.  Perhaps  our 
conception  of  immunity  in  the  past  has  been  influenced  too 
strongly  by  a  study  of  these  latter  conditions,  which  are  readily 
induced  in  the  laboratory,  but  rarely  if  ever  seen  in  the  actual 
practice  of  medicine.  They  represent  in  an  extreme  form  the 
changes  which  follow  disease  of  natural  origin,  and  possess  the 
theoretical  interest  which  attaches  to  all  extreme  cases. 

ACQUIRED  IMMUNITY  occurs  in  two  distinct  forms — active  and 
passive.  A  third  form  exists,  which  we  may  call  mixed,  since  it  is 
brought  about  by  a  combination  of  the  procedures  necessary  for 
the  development  of  the  other  two. 

Active  immunity  may  be  defined  as  acquired  immunity,  due  to 
an  attack  of  the  disease  in  question  in  its  normal  form,  or  in  a 
modified  and  less  severe  form  of  artificial  production.  The 
essential  feature  is  that  the  cells  and  tissues  of  the  person  or 
animal  should  be  subjected  to  the  action  of  the  bacterium  (or  its 
toxin),  and  by  its  own  efforts,  and  as  a  result  of  an  active  struggle 
with  it,  should  become  less  susceptible  to  its  toxin  than  before. 
Active  immunity  is  developed  only  as  a  result  of  an  illness  of  the 
host,  due  to  the  action  of  the  microbe  on  its  cells  ;  and  this  illness 
may  be  of  any  degree  of  severity,  ranging  from  an  unmodified 
attack  of  the  disease  which  may  threaten  life  down  to  the  most 
transitory  and  unimportant  reaction  due  to  an  injection  of  a 
minute  dose  of  a  mild  vaccine.  And  one  of  the  great  aims  of 
modern  preventive  medicine  is  to  reduce  the  severity  of  the  disease 
necessary  to  produce  acquired  immunity  to  a  minimum.  The 
greatest  step  ever  made  in  this  direction  was  Jenner's  substitution 
of  vaccination  for  inoculation.  In  each  case  the  effect  is  the  same 
as  regards  the  resulting  immunity  (though  in  different  degree), 
but  the  disease  in  the  former  case  is  mild  and  devoid  of  danger, 
in  the  latter  severe  and  dangerous.  As  a  general  rule,  it  may  be 
taken  that  the  severer  the  disease  the  stronger  and  more  lasting 
the  acquired  immunity.  A  good  example  will  be  quoted  when 
dealing  with  mixed  immunity.  This  is  not  necessarily  the  case, 


INTRODUCTORY   AND    GENERAL  21 

however,  for  the  repeated  injections  of  vaccines  which  are  so  mild 
as  not  to  cause  any  noticeable  general  and  very  little  local  reaction 
may  induce  a  high  degree  of  immunity. 

The  main  methods  in  which  active  immunity  is  acquired  are 
these : 

i.  A  natural  attack  of  the  disease,  or  an  attack  which  is  natural 
in  course,  but  of  artificial  induction.  The  only  example  of  the 
latter  in  human  medicine  is  the  now  disused  practice  of  smallpox 
inoculation,  in  which  the  person  to  be  protected  was  inoculated 
with  the  disease,  which  ran  a  perfectly  usual  course,  and  was  not 
infrequently  fatal.  As  a  rule,  however,  it  was  milder  than  the 
naturally  acquired  smallpox,  since  the  infective  material  was 
taken  from  a  favourable  case,  and  the  operation  performed  when 
the  patient  was  in  good  health  and  able  to  get  proper  attention 
from  the  outset.  Probably,  too,  the  severity  of  the  disease  was 
somewhat  modified  by  the  fact  that  the  virus  did  not  reach  the 
body  by  the  usual  route.  But  the  infection  was  ordinary  small- 
pox, and  might  start  an  ordinary  epidemic. 

The  process  is  used  to  a  much  greater  extent  in  veterinary 
practice,  where  an  occasional  death  due  to  the  induced  disease  is 
of  comparatively  little  importance  if  thereby  the  outbreak  can  be 
controlled  or  the  great  majority  of  the  flock  saved.  As  a  rule, 
an  attempt  is  made  to  render  the  attack  as  mild  as  possible, 
either  by  (a)  limiting  the  amount  of  the  infective  material  used, 
or  (b)  by  introducing  it  in  an  abnormal  way,  or  (c)  inoculating 
animals  at  a  time  when  they  are  found  to  be  least  susceptible,  or 
by  a  combination  of  these  methods.  Thus  Texas  fever  is  a 
disease  of  cattle  due  to  a  protozoon  (Piroplasma  bigemimim)  which 
is  conveyed  by  the  bites  of  ticks.  One  of  the  methods  used  for 
the  protection  of  cattle  in  infected  districts  is  to  expose  calves 
whilst  still  milk-fed  to  the  bites  of  a  few  infected  ticks ;  another 
is  to  inject  blood  from  diseased  animals  (containing  the  parasite) 
in  small  doses  direct  into  the  jugular  vein.  In  favourable  cases 
the  result  is  a  severe  attack  of  the  disease,  which,  however,  is 
rarely  fatal,  and  is  followed  after  a  time  by  complete  immunity. 
In  some  cases  the  disease  is  but  slight,  and  in  them  a  second  or 
even  third  dose,  in  each  case  larger  than  the  preceding,  is  required. 
The  mortality  from  the  injections  is  from  3  to  10  per  cent.,  whilst 
that  of  untreated  animals  in  infected  areas  is  about  90  per  cent. 

A  similar  method  is  in  use  for  combating  rinderpest,  but  here 
bile  from  an  animal  dead  of  the  disease  is  used  as  the  infecting 


22  METHODS    OF   VACCINATION 

agent,  since  the  blood  frequently  contains  other  infective  materials 
which  would  complicate  the  issue. 

In  pleuro-pneumonia  of  cattle  the  severity  of  the  disease  is 
lowered  by  altering  the  route  of  infection.  In  the  natural  disease 
the  infection  probably  enters  by  the  lung,  and  its  course  is  severe 
and  dangerous.  Protection  is  conferred  by  inoculating  virus  from 
the  lung  of  an  animal  dead  of  the  disease  into  the  subcutaneous 
tissue  near  the  tail ;  much  local  swelling  results,  and  general 
immunity  is  established.  Perhaps,  strictly  speaking,  this  method 
of  induction  of  active  immunity  should  be  put  in  a  class  of  its 
own ;  it  is  one  in  which  a  local  is  substituted  for  a  general  disease, 
with  the  obvious  result  of  greatly  lessening  its  severity. 

The  material  used  in  the  production  of  artificial  immunity  of 
the  type  we  are  describing  is  sometimes  called  a  vaccine.  This 
is  undesirable,  and  it  is  advisable  to  use  the  word  virus  for  material 
containing  the  infective  agent  in  its  normal  virulence,  retaining 
the  word  vaccine  for  that  in  which  the  bacterium  has  entirely  lost 
its  power  of  producing  the  normal  disease,  whatever  the  dose  and 
whatever  the  channel  of  introduction.  The  term  is  a  somewhat 
unfortunate  one  etymologically,  but  it  is  in  such  general  use  that 
it  is  hopeless  to  attempt  to  displace  it. 

2.  By  the  use  of  living  cultures  of  pathogenic  bacteria  of 
diminished  or  altered  virulence — i.e.,  of  a  living  vaccine.  There 
are  as  many  modifications  of  this  method  as  there  are  ways  of 
mitigating  the  virulence  of  a  culture,  and  different  methods  are 
applicable  to  different  diseases. 

(a)  By  the  use  of  vaccines  diminished  in  virulence  by  passage 
through  animals.     The  most  important  example  of  this  is,   of 
course,   Jennerian   vaccination.      It   would   take   us   too   far   to 
examine  the  evidence  in  favour  of  this  view,  but  it  may  be  taken 
as  fairly  proved  that  ordinary  lymph  vaccine  consists  of  a  culture 
of  the  smallpox  organism  modified  by  passage  through "  calves, 
the  modification  being  of  such  a  nature  that  it  has  lost  its  power 
of  producing  a  general  disease  (smallpox),  but  retained  that  of 
causing  a  local  one  (vaccinia)  otherwise  similar  in  nature. 

We  have  already  referred  to  the  decrease  in  the  virulence  of  the 
bacillus  of  swine  erysipelas  on  passage  through  rabbits,  and  the 
use  of  these  mitigated  cultures  as  a  vaccine  for  pigs.  This  is  a 
better  example  of  the  type  of  immunity  we  are  considering,  since 
it  has  to  do  with  a  known  organism. 

(b)  By  the  use  of  vaccines  in  which  the  virulence  is  diminished 


INTRODUCTORY   AND    GENERAL  23 

by  drying.  The  only  practical  example  is  rabies.  There  the 
process  of  immunization  is  carried  out  by  means  of  the  use  of  a 
series  of  vaccines  of  gradually  increasing  degrees  of  virulence,  the 
degree  dependent  on  the  time  for  which  drying  has  gone  on.  It 
is,  of  course,  necessary  to  proceed  with  extreme  caution,  since  the 
cords  that  have  been  dried  for  but  a  few  days  are  still  infective 
and  virulent,  and  the  amount  of  natural  immunity  in  man  is 
extremely  small,  so  that  an  attempt  to  accelerate  the  process 
might  be  fatal.  The  method  varies  somewhat  at  different  labora- 
tories, but  the  following  may  be  taken  as  a  type  of  the  procedure 
used.  It  is  of  interest  as  being  the  method  used  in  the  first  case 
treated — that  of  Joseph  Meister. 

Day  i. — Inoculation  with  vaccine  made  by  drying  the  cord  for 
fourteen  days.  A  second  injection  with  cord  treated  for  ten 
days. 

Day    2. — Two  injections  ;  cords  dried  for  eleven  and  nine  days. 

Day    3. — One  injection  ;  cord  dried  for  eight  days. 

Day    4. — One  injection ;  cord  dried  for  seven  days. 

Day    5. — One  injection  ;  cord  dried  for  six  days. 

Day    6. — One  injection  ;  cord  dried  for  five  days. 

Day    7. — One  injection  ;  cord  dried  for  four  days. 

Day    8. — One  injection  ;  cord  dried  for  three  days. 

Day    g. — One  injection  ;  cord  dried  for  two  days. 

Day  10. — One  injection ;  cord  from  a  rabbit  which  had  died 
the  same  day,  and  which  was  therefore  unaltered  in  virulence. 

The  method  in  use  in  France  at  the  present  day  is  almost  like 
this,  except  that  the  latter  stages  are  repeated  twice,  or,  in  severe 
cases,  three  times — i.e.,  on  the  ninth  and  fourteenth  days  in  mild 
cases  (and  on  the  nineteenth  also  in  severe  ones)  injections  of 
nine-day  cords  are  started,  and  the  strength  increased  rapidly,  so 
that  three-day  cords  are  used  on  the  thirteenth,  eighteenth,  and 
twenty-first.  In  Germany  the  treatment  is  begun  with  eight-day 
cords,  the  older  ones  being  considered  inert. 

(c)  By  the  injection  of  living  cultures  modified  by  heat.     The 
classical  example   is  vaccination   against  anthrax  by  means  of 
Pasteur's  two  vaccines,  the  method  of  preparing  which  is  given 
on  p.  1 8.     The  first  vaccine  is  injected,  and  is  followed  by  the 
second  in  about  a  fortnight,  immunity  being  established  in  about 
another  fortnight. 

(d)  By  the  injection  of  cultures  attenuated  by  prolonged  cultiva- 
tion in  vitro.     The  use  of  this  method  in  the  case  of  chicken 


24  VACCINES   OF   DEAD    BACTERIA 

cholera  has  been  referred  to  already,  and  it  is  the  one  usually 
employed  in  the  laboratory,  where  old  cultures  are  used  in  pre- 
ference to  more  virulent  ones  in  the  early  stages  of  immunization. 

(e)  By  the  use  of  very  small  doses  of  living  cultures  of  full 
virulence.  This  has  been  proved  possible  in  anthrax,  symptomatic 
anthrax,  and  some  other  diseases.  At  present  the  process  is  more 
interesting  than  practically  useful,  but  it  has  been  used  clinically 
in  the  case  of  tubercle,  treatment  being  commenced  by  the  injec- 
tion of  a  single  living  bacillus,  and  promising  results  have  been 
obtained. 

3;  A  third  class  of  methods  consists  in  the  use  of  vaccines 
composed  of  dead  bacteria.  The  advantages  are  obvious  :  the 
dose  is  under  accurate  control ;  the  disease  which  it  induces  is 
self -limited,  so  that  it  is  impossible  for  a  general  infective  process 
to  be  produced  when  used  on  a  person  of  deficient  natural 
immunity ;  and  the  vaccine  is  easy  to  keep  in  a  condition  ready 
for  immediate  use.  Hence  this  method  is  mostly  used  in  human 
medicine,  whereas  the  use  of  mitigated  or  unmitigated  viruses  is 
mainly  confined  to  veterinary  work.  The  methods  used  in  the 
preparation  of  the  vaccines  varies  greatly  in  the  different  cases, 
and  here  we  can  only  glance  at  some  of  the  general  principles. 
In  preparing  the  cultures,  the  most  careful  precautions  have  to  be 
taken  to  insure  the  purity  of  the  microbe  used  and  absence  of  all 
other  pathogenic  forms,  especially  perhaps  the  spores  of  the 
tetanus  bacillus.  The  age  of  the  culture  has  to  be  determined  by 
the  necessities  of  the  case,  but  as  a  rule  young  cultures  are 
preferable.  The  method  by  which  the  bacteria  is  killed  also 
varies,  but  heat  is  generally  employed,  and  as  a  rule  the  shorter 
the  exposure  and  the  lower  the  temperature  the  better.  In  other 
cases  the  bacteria  are  emulsified  in  saline  solution  and  allowed  to 
undergo  autolysis  at  the  body  temperature,  sterility  being  ensured 
subsequently  by  means  of  heat  or  chemical  antiseptics ;  or  they 
may  be  killed  with  a  minimum  of  heat,  and  submitted  to  autolysis 
at  37°  C.  subsequently. 

There  are  numerous  methods  of  determining  the  dose  to  be 
used,  (a)  A  definite  fraction  of  an  agar  or  other  culture  of 
known  age  may  be  taken,  or,  what  comes  to  much  the  same 
thing,  the  growth  from  so  many  square  centimetres  or  millimetres 
of  surface  of  the  culture  medium.  (b)  The  amount  may  be 
judged  by  the  weight,  and  this  is  the  method  used  in  the  case  of 
tubercle.  When  it  is  employed  with  other  bacteria  it  is  usually 


INTRODUCTORY   AND    GENERAL  25 

carried  out  by  means  of  standard  loops,  each  of  which  will  pick 
up  a  known  amount  of  growth.  (c)  In  Wright's  ingenious 
method  of  counting  a  vaccine  a  certain  amount  of  the  latter  is 
mixed  with  human  blood  in  definite  proportions,  and  films  are 
prepared  and  stained.  The  numbers  of  red  corpuscles  and  of 
bacteria  in  several  fields  of  the  microscope  are  then  counted,  and 
(the  numbers  of  red  corpuscles  in  a  definite  volume  of  the  blood 
being  known)  the  proportions  of  the  two  will  permit  of  the  calcu- 
1  ation  of  the  numbers  of  the  bacteria,  (d)  Some  determine  the 
strength  of  the  vaccine  by  reference  to  a  permanent  standard, 
usually  consisting  of  a  fine  suspension  of  barium  sulphate.  A 
strong  emulsion  of  bacteria  is  prepared  and  diluted  until  it 
matches  the  standard,  (e)  The  volume  of  the  bacteria  in  the 
emulsion  may  be  determined  by  centrifugalization  in  a  graduated 
tube,  and  a  certain  volume  of  sediment  made  up  to  a  certain 
volume  of  vaccine.  (/)  The  number  of  bacteria  present  in  the 
emulsion  may  be  counted  directly  by  the  use  of  the  counting 
chamber  of  the  hsemocytometer,  and  this  is  the  method  I  usually 
employ.  The  emulsion  is  diluted  (usually  to  twenty  times  its 
volume)  with  a  dilute  solution  of  methylene  blue  or  other  stain, 
boiled,  and  a  drop  placed  in  the  counting  chamber  and  prepared  as 
if  it  were  a  blood  specimen  in  which  the  red  corpuscles  were  to  be 
counted,  A  ^-inch  lens  and  a  high  eyepiece  are  used,  and,  as  a 
rule,  the  process  presents  no  difficulty. 

In  all  cases  an  addition  of  a  chemical  antiseptic  is  advisable 
to  avoid  subsequent  contamination.  Carbolic  acid  or  lysol  (0*25  to 
0-5  per  cent.)  are  most  used;  another  good  plan  is  to  keep  a  few 
drops  of  chloroform  at  the  bottom  of  the  bottle,  so  that  the  fluid 
is  always  saturated. 

This  method  is  mostly  used  in  plague,  cholera,  and  enteric 
fever  in  preventive  medicine,  and  in  the  treatment  of  infective 
processes  by  Sir  Almroth  Wright's  method  in  curative  medicine. 
These  will  be  discussed  subsequently. 

4.  Inoculation  with  the  chemical  products  or  with  the  toxins 
of  the  bacteria,  the  bodies  of  the  bacteria  themselves  being 
removed  by  filtration  or  in  some  other  way.  This  is  obviously 
closely  allied  to  the  last  method — the  use  of  killed  cultures. 

It  was  introduced  by  Smith  and  Salmon  in  the  case  of  hog 
cholera,  and  is  now  chiefly  used  in  the  immunization  of  animals 
for  the  production  of  antitoxic  sera.  It  is  considered  fully  in  a 
subsequent  chapter. 


26  PASSIVE    IMMUNITY 

PASSIVE  IMMUNITY,  the  second  form  of  acquired  immunity,  is 
conferred  by  injecting  into  a  susceptible  animal  the  serum  of  one 
which  has  acquired,  an  active  immunity  to  the  disease  in  question. 
It  is  a  kind  of  second-hand  immunity,  acquired  in  virtue  of  the 
reception  of  substances  actively  formed  by  another  animal  which 
has  had  to  fight  against  the  infecting  agent  in  order  to  form  them. 
In  its  production  there  is  no  necessary  illness,  however  slight. 
Such  may  occur,  it  is  true,  but  it  is  not  more  than  would  be  pro- 
duced by  normal  serum,  and  stands  in  no  necessary  relationship 
to  the  development  of  the  immunity.  And  when  such  illness  does 
occur,  it  does  so  after  the  production  of  the  immunity,  and  may 
be  very  severe  when  the  protection  given  is  but  slight,  and 
vice  versa. 

For  the  production  of  passive  immunity  it  is  necessary  to 
inject  the  serum  of  an  animal  which  has  been  artificially  immun- 
ized, that  from  one  which  is  naturally  immune  being  devoid 
of  action  in  this  respect.  To  this  general  rule  there  are 
one  or  two  exceptions,  which  are  perhaps  more  apparent  than 
real. 

Passive  immunity  is  sometimes  called  antitoxic.  The  term, 
however,  is  not  a  good  one,  since  there  are  several  varieties  of 
passive  immunity,  only  one  of  which  is  due  to  an  antitoxin. 

Passive  immunity  is  specific — that  is,  the  serum  of  an  animal 
which  has  acquired  immunity  against  one  organism  will  protect 
a  second  against  that,  and  against  no  other.  In  this,  of  course, 
it  resembles  active  immunity,  but  the  two  differ  in  several 
important  particulars. 

i.  As  regards  its  production.  Active  immunity  takes  some 
time— usually  a  week  or  so — to  develop,  dating  from  the  infection 
or  injection  of  the  vaccine,  and  in  many  cases  at  least  its 
appearance  is  preceded  by  a  negative  phase,  in  which  the  natural 
immunity  to  the  organism  in  question  is  lowered.  But  passive 
immunity  is  established  as  soon  as  the  serum  has  become  mixed 
with  the  blood  of  the  person  or  animal  injected,  and  there  is  no 
negative  phase. 

Hence  in  severe  infections  our  best  hope  in  the  way  of  specific 
medication  is  in  the  production  of  passive  immunity.  It  is  but 
recently  that  the  injection  of  vaccines  was  thought  of  in  face  of 
an  infection  already  developed,  and  it  is  obvious  that  the  method 
will  be  useless  or  dangerous  in  very  severe  and  rapid  infective 
processes.  Passive  immunity,  on  the  other  hand,  can  be  induced 


INTRODUCTORY   AND    GENERAL  27 

at  once  and  without  a  negative  phase.     Unfortunately,  it  is  not 
always  or  often  possible. 

2.  As  regards  its  duration.  Active  immunity  lasts  for  a  long 
time,  the  length  differing  greatly  in  different  diseases  and  after 
various  methods  of  induction.  In  many  cases  it  lasts  a  year  or 
more.  Passive  immunity,  on  the  other  hand,  is  always  of  brief 
duration,  and  lasts  only  about  as  long  as  the  serum  injected  is 


FIG.  2.— SHOWING  THE  SEQUENCE  OF  EVENTS  IN  THE  PRODUCTION  OF 
ACTIVE  IMMUNITY. 

An  injection  of  vaccine  at  b  is  followed  by  a  decrease  in  the  degree  of 
immunity  (negative  phase),  a  rise,  and  a  gradual  return  to  the  normal 
condition. 

actually  present  in  the  blood.  It  depends  to  a  certain  extent  on 
the  dose  of  serum  given,  and  also  on  the  species  of  animal  from 
which  it  was  derived.  An  animal  of  a  certain  species  is  immu- 
nized for  a  longer  period  by  serum  from  another  animal  of  the 
same  kind  than  from  one  of  a  different  species.  In  general  terms 
the  duration  of  passive  immunity  is  three  to  six  weeks.  It  is 
renewable  at  pleasure,  as  far  as  we  know  indefinitely. 


a 


FIG.  3. — SHOWING  THE  SEQUENCE  OF  EVENTS  IN  PASSIVE  IMMUNITY. 
An  injection  of  serum  is  given  at  b. 

Hence  passive  immunity  is  chiefly  of  value  to  ward  off*  an 
infection  the  danger  to  which  is  of  short  duration.  Thus  in 
veterinary  practice  the  passive  immunity  of  horses  conferred  by 
the  injection  of  tetanus  antitoxin  is  of  the  greatest  possible  value 
before  operations,  or  immediately  after  the  infliction  of  a  wound, 
horses  being  so  prone  to  tetanus  that  in  some  places  any  opera- 


28  MIXED   IMMUNITY 

tion  was  a  matter  of  great  danger  before  the  introduction  of  this 
method. 

Passive  immunity  is  also  useful  as  a  basis  for  active  immunity 
This  will  be  described  under  the  heading  of  Mixed  Immunity. 

3.  Passive   immunity   for  a  given    bacterium  or  its   products 
cannot  be  made  so  potent  as  the  active  form  for  the  same  disease 
in  the  same  animal  species.     The  reason  is  obvious :  the  passive 
form  only  occurs  in  virtue  of  the  presence  in  the  blood  of  some  of 
the  foreign  serum,  which  can  never  form  more  than  a  fraction  of 
the  whole  fluid.  The  degree  of  the  immunity  may  be  sufficient  for 
all  practical  purposes,  but  can  never  reach  the  enormous  height 
met  with  in  hypervaccinated  animals. 

4.  Active  immunity  we  believe  to  be  developed  to  some  extent 
in  all,  or  almost  all,  infections,  but   the  production  of  passive 
immunity  is  impossible  in  very  many  cases — e.g.,  tubercle  (as  far 
as  we  know  at  present),  infections  with  pyocyaneus,  glanders, 
malaria,  and  many  other  parasitic  organisms.     Perhaps  in  the 
future  we  shall  be  able  to  procure  active  sera  against  all  organisms, 
but  at  present  we  have  comparatively  few  of  any  value. 

MIXED  IMMUNITY  is  a  combination  of  the  two  forms  already 
described,  in  which  the  dangers  and  delay  incidental  to  the  induc- 
tion of  active  immunity  are  avoided  by  the  use  of  a  protective 
serum.  It  is  really  a  succession  of  the  two  forms,  the  passive 
immunity  being  developed  at  once  as  a  consequence  of  the  injec- 
tion of  the  serum,  whilst  the  active  form  develops  later  in  conse- 
quence of  the  vaccination.  The  process  is  sometimes  called 
sero- vaccination. 

It  is  not  of  great  importance  in  human  pathology,  the  chief 
example  being  the  form  of  typhoid  inoculation  suggested  by 
Besredka,  and  not  yet  used  on  a  large  scale.  In  it  the  killed 
typhoid  bacilli  are  submitted  to  the  action  of  the  immune  serum, 
from  which  they  absorb  certain  protective  substances  and  become 
modified  thereby.  It  is  claimed  that  this  treatment  prevents  the 
development  of  the  discomfort  that  follows  the  use  of  ordinary 
typhoid  vaccine,  and  that  the  immunity  is  developed  very  rapidly. 
It  may  be  followed  by  an  injection  of  ordinary  vaccine. 

The  method  is  used  to  a  considerable  extent  by  veterinary 
surgeons,  and  there  are  several  modifications  in  the  process,  the 
serum  being  injected  either  mixed  with  the  virus,  or  before,  or 
after,  or  simultaneously  in  different  sides  of  the  body.  Thus  in 
the  treatment  of  South  African  horse-sickness  the  virus  (the  blood 


INTRODUCTORY   AND   GENERAL  29 

of  diseased  animals)  may  be  mixed  with  the  serum  from  hyper- 
immunized  animals  and  injected  subcutaneously.  If  the  serum 
and  virus  are  injected  separately  the  animal  will  in  all  cases 
acquire  passive  immunity ;  but  unless  there  is  some  degree  of  ill- 
ness (a  " reaction")  this  will  be  but  temporary,  and  no  active 
immunity  will  be  superadded.  Thus,  if  the  serum  be  injected  and 
the  virus  given  subcutaneously  at  the  same  time,  no  reaction 
follows,  and  the  immunity  does  not  last  more  than  a  month  ;  but 
if  the  injection  is  made  into  a  vein  a  reaction  occurs,  and  active 
immunity,  lasting  for  about  a  year,  will  follow  (Stockman). 

The  method  is  also  used  in  the  early  stages  of  antitoxin  forma- 
tion, the  horse  being  treated  with  a  mixture  of  toxin  and  anti- 
toxin, the  latter  being  in  excess.  But  here  it  seems  unquestionable 
that  active  immunity  is  acquired,  and  the  mechanism  by  which 
this  occurs  is  discussed  subsequently. 


FIG.  4. — MIXED  IMMUNITY. 
The  presence  of  a  negative  phase,  as  shown  in  the  diagram,  is  not  essential. 

LOCAL  IMMUNITY.— We  have  hitherto  spoken  of  the  body  as  a 
whole,  assuming  that  all  parts  are  equally  resistant  or  susceptible. 
This  is  not  the  case,  and  certain  parts  are  found  to  have  a  marked 
degree  of  immunity  to  certain  bacteria.  Here  we  have  to  be  sure 
that  we  are  dealing  with  regions  that  are  equally  exposed  to 
infection.  The  stomach,  for  example,  is  comparatively  rarely 
attacked  by  infective  processes,  and  this  may  be  due  to  the  fact 
that  the  gastric  juice  is  of  a  sufficient  degree  of  acidity  to  kill  or 
inhibit  most  bacteria.  Yet  here  it  is  probable  that  this  does  not 
account  for  all  the  phenomena,  and  that  some  degree  of  true  local 
immunity  does  exist.  Numerous  other  examples  may  be  quoted. 
Pneumococcic  infections  are  common  in  the  lungs  and  pleura,  but 
rarely  spread  further,  and  cause  disease  of  the  ribs  and  intercostal 
muscles ;  tubercle  is  common  in  the  bones  and  extremely  rare  in 
the  muscles,  whilst  Trichina  spimlis  affects  the  muscles  and  never 


30  LOCAL    IMMUNITY 

attacks  the  bones,  and  rarely  any  other  tissues.  Some  of  the  best 
examples  may  be  taken  from  diseases  that  spread  by  continuity 
from  one  tissue  to  another.  Thus  the  gonococcus  in  either  sex 
spreads  along  the  urethra  with  ease,  but  seldom  involves  the 
mucous  membrane  of  the  bladder ;  it  practically  never  attacks  the 
vaginal  mucosa  (in  adults),  but  spreads  from  the  cervical  to  the 
corporeal  endometrium,  and  thence  to  the  Fallopian  tubes,  but 
comparatively  rarely  goes  farther  and  produces  a  general  peri- 
tonitis. Diphtheria,  too,  though  it  may  spread  in  any  direction, 
seldom  creeps  down  the  oesophagus.  Many  other  examples  might 
be  quoted. 

There  are  marked  differences  in  regard  to  local  immunity 
between  the  child  and  the  adult.  The  most  marked  example, 
perhaps,  is  in  the  almost  perfect  local  immunity  of  the  scalp  to 
ringworm  in  adults,  which  contrasts  so  markedly  with  the  absolute 
susceptibility  of  children,  whereas  the  susceptibility  of  the  skin  of 
the  body  to  the  same  parasite  is,  if  anything,  greater  in  the  former. 
In  most  cases  of  differing  immunity  at  different  ages  the  child  is 
more  susceptible,  just  as  its  resistance  to  general  diseases  is  less, 
and  the  few  exceptions  that  may  be  quoted  are  perhaps  rather 
apparent  than  real. 

Local  immunity  may  be  natural  or  acquired.  Passive  im- 
munity, of  course,  cannot  be  local  for  long,  as  any  serum  which 
is  injected  will  rapidly  diffuse  away  and  be  removed  by  the 
lymphatics  and  blood-stream.  The  cases  mentioned  above  are 
all  examples  of  natural  local  immunity.  The  difference  between 
the  reactions  of  the  tissues  of  children  and  adults  do  not  neces- 
sarily point  to  the  acquisition  of  any  active  immunity  in  the  sense 
in  which  the  word  has  been  defined  above,  but  rather  to  the 
general  rise  in  resisting  power  accompanying  the  general  improve- 
ment in  strength  and  vitality,  and  in  some  cases,  perhaps,  to  an 
actual  maturation  of  the  tissues,  as  in  the  case  of  the  adult  vaginal 
mucous  membrane,  which  is  immune  to  the  gonococcus,  whereas 
the  thin  and  immature  infantile  membrane  is  susceptible.  The 
immunity  of  the  adult  scalp  to  ringworm  also  is  not  acquired, 
using  the  word  in  the  narrow  sense,  for  it  occurs  apart  altogether 
from  an  attack  of  the  disease. 

Our  knowledge  of  acquired  local  immunity  is  very  incomplete ; 
it  is  a  difficult  subject  for  research,  and  more  attention  has  been 
paid  to  general  immunity.  A  little  consideration  will  demonstrate 
the  fact  of  its  occurrence.  For  example,  when  a  person  develops 


INTRODUCTORY   AND    GENERAL  3! 

crops  of  boils  it  will  often  be  found  that  one  is  undergoing  involu- 
tion whilst  another  is  developing ;  hence  the  cure  of  the  first 
cannot  be  due  to  any  general  immunity,  but  must  depend  on  local 
changes  which  do  not  affect  the  second.  A  similar  line  of  argu- 
ment will  show  the  development  of  acquired  immunity  to  the 
streptococcus  in  erysipelas  ;  the  healthy  skin  is  susceptible,  since 
the  disease  spreads  to  it,  but  the  process  does  not  extend  back- 
ward into  an  area  already  affected,  but  now  cured,  or  does  so  but 
rarely. 

The  subject  cannot  be  discussed  further  with  advantage,  and 
will  be  deferred  to  a  subsequent  chapter,  when  the  known  factors 
on  which  immunity  depends  have  been  elucidated. 

There  are  important  non-specific  causes  for  alterations  in  local 
immunity,  as  is  the  case  with  general.  These  practically  resolve 
themselves  into  the  presence  or  absence  of  an  adequate  supply  of 
blood  ;  the  more  copious  the  supply  of  healthy  circulating  blood, 
the  greater  the  resistance  to  infections,  and  vice  versa.  Hence  the 
utility  of  fomentations  and  other  hot  applications  in  the  initial 
stages  of  an  infective  lesion  ;  hence,  too,  the  application  of  Bier's 
method  of  passive  congestion,  in  which  an  excess  of  blood 
(though  partly  stagnant)  is  made  to  flush  the  tissues.  And  there 
is  no  doubt  that  the  object  of  the  dilatation  of  the  vessels  and 
acceleration  of  the  flow  of  blood  through  them  which  occurs  in 
the  early  stages  of  inflammation  is  a  beneficial  process  which 
has  this  improvement  of  the  local  resisting  powers  as  one  of  its 
objects,  the  influx  of  an  increased  number  of  leucocytes  and  the 
dilution  and  removal  of  the  soluble  toxins  being  others.  In  acute 
inflammation  we  may  distinguish  two  stages.  In  the  first,  the 
stage  just  mentioned,  the  conservative  reaction  of  the  vessels  is 
most  obvious,  and  in  the  case  of  a  mild  infection,  or  if  the 
immunity  is  very  strong,  may  suffice  to  destroy  and  remove  the 
infective  material  and  its  toxin.  The  stagnation  and  ultimate 
cessation  of  the  blood-flow  are  indications  that  the  irritant  is, 
temporarily  at  least,  getting  the  upper  hand,  and,  by  cutting  off 
the  blood-supply,  is  neutralizing  the  most  powerful  defensive 
factor.  The  acceleration  of  the  flow  may  be  regarded  as  physio- 
logical, the  retardation  and  cessation  as  pathological. 

The  causes  of  local  reduction  of  immunity  by  obstruction  of  the 
blood-stream  are  numerous,  the  most  important  being  traumatism 
(by  injuring  the  vessels  going  to  the  region),  endarteritis,  throm- 
bosis, tight  bandaging,  etc.  They  need  not  be  discussed  at 


32  LOCAL    IMMUNITY 

length,  but  it  is  advisable  to  point  out  that  severe  traumatism,  in 
the  form  of  violent  laceration  and  contusion  of  a  part,  is  an 
extremely  powerful  predisposing  agent,  and  that  it  acts  in  two 
ways,  or  perhaps  more.  In  the  first  place,  there  may  be  some 
death  of  tissues,  either  in  small  or  large  amounts,  and  in  these 
dead  tissues  the  natural  resisting  powers  are  of  course  in  abeyance, 
so  that  the  bacteria  will  grow  unchecked,  as  they  would  in  dead 
culture  media ;  and,  secondly,  that  the  blood  does  not  reach  this 
dead  material,  and  the  leucocytes  only  do  so  with  difficulty.  The 
importance  of  this  is  well  seen  in  tetanus.  The  normal  tissues 
have  a  considerable  degree  of  resistance  to  this  organism,  and 
infection  rarely  takes  place  in  a  clean  incised  wound,  even  in 
cases  in  which  we  can  be  almost  certain  that  the  spores  of  the 
tetanus, bacillus  have  been  introduced. 

Another  cause  of  reduced  local  immunity  is  the  action  of 
irritants  on  the  tissues.  Here  we  must  distinguish  two  cases. 
If  the  irritant  be  but  mild,  it  may  be  actually  beneficial ;  it 
causes  the  earlier  phenomena  of  inflammation  which  we  have 
previously  referred  to  as  being  protective,  and  may  tend  to  raise 
the  resistance  of  the  part  in  consequence.  Thus,  according  to 
many  observers  (who  do  not  agree  precisely  on  the  interpretation), 
the  injection  of  a  small  quantity  of  almost  any  bland  (but  never- 
theless foreign)  substance  into  the  peritoneal  cavity  may  protect 
an  animal  against  a  lethal  dose  of  a  bacterial  culture  introduced 
subsequently;  normal  saline  solution,  water,  broth,  serum,  etc., 
all  have  this  action.  But  if  the  irritant  be  more  powerful,  so  that 
the  tissues  are  killed  and  the  vessels  occluded,  or  the  leucocytes 
killed,  the  susceptibility  of  the  region  is  greatly  increased. 
Chemical  antiseptics  have  this  action,  especially  in  certain 
regions,  such  as  the  peritoneum.  The  same  thing  may  be  demon- 
strated experimentally.  Tetanus  spores  washed  free  of  toxin 
will  not  produce  tetanus  in  rabbits,  but  will  do  so  if  an  irritant 
such  as  lactic  or  carbolic  acid  is  injected  simultaneously. 

A  few  words  may  be  said  here  on  the  phenomena  of  immunity 
arid  susceptibility  in  relation  to  the  modifications  they  cause  in 
the  infective  processes.  Where  the  immunity  is  great,  or,  as  we 
say,  absolute,  the  result  of  an  injection  of  the  infective  agent  is 
nil ;  there  is,  of  course,  some  degree  of  inflammation,  but  this 
follows  the  injection  of  any  fluid,  even  normal  saline  solution,  and 
the  effect  of  the  bacteria  themselves  is  inappreciable.  In  this 
case,  therefore,  the  bacteria  are  immediately  destroyed,  and  the 


INTRODUCTORY   AND    GENERAL  33 

substances  which  they  produce  are  without  deleterious  effect  on 
the  cells  of  the  body.  In  another  group  of  cases,  referred  to 
above,  the  bacteria  do  not  die,  but  their  toxins  remain  harmless 
to  the  host ;  this  is  Ehrlich's  immunitas  non  stevilisans,  and  it  occurs 
in  the  case  of  many  of  the  lower  animals  which  have  in  their  blood 
various  protozoa  (trypanosomes,  etc.),  without  thereby  suffering 
the  slightest  appreciable  injury.  In  man  the  condition  is  best 
seen  in  its  acquired  form  in  the  immunity  possessed  by  negroes 
to  the  action  of  malaria  parasites,  though  the  plasmodium  may  be 
found  in  the  blood.  A  closely  allied  phenomenon  is  in  the  latency 
of  bacteria.  Thus  a  person  may  develop  an  attack  of  typhoid 
osteitis  years  after  an  attack  of  typhoid  fever,  and  we  can  only 
assume  that  the  bacteria  have  lain  latent  in  his  tissues  for  this 
time ;  in  all  probability  they  have  been  kept  from  infecting  him  as 
a  result  of  a  sufficient  degree  of  immunity,  and  when  this  breaks 
down  or  wears  off  a  renewed  outburst  occurs.  The  gonococcus 
may  be  latent  in  a  similar  way  for  periods  equally  long.  Another 
similar  phenomenon  is  the  carriage  of  infection  by  persons  who 
remain  themselves  healthy.  Diphtheria  is  a  common  example, 
and  it  is  no  rarity  to  find  a  person  in  whose  throat  diphtheria 
bacilli  are  present,  but  who  remains  unattacked.  Here  the 
immunity  suffices  to  prevent  the  bacillus  from  invading  the  body, 
but  not  to  destroy  it. 

At  the  opposite  end  of  the  scale  occur  those  cases  in  which 
immunity  is  practically  absent.  Here  the  result  of  the  introduc- 
tion of  the  bacteria  is  a  rapid  infection,  both  local  and  general, 
with  profound  symptoms  of  intoxication ;  the  bacteria  spread 
through  the  tissues  just  as  they  would  through  a  good  culture 
medium,  and,  in  addition,  invade  the  blood  and  multiply  therein. 
This  is  rarely  seen  in  man,  though  some  examples  of  septicaemic 
plague  and  streptococcal  septicaemia  from  post-mortem  wounds 
approach  it  closely.  It  can  be  produced  experimentally  in 
animals,  when  large  doses  of  virulent  cultures  are  injected. 
Death  follows  in  a  few  hours,  and  the  blood  is  found  to  be  swarm- 
ing with  bacteria. 

Between  these  two  extremes  come  those  cases  in  which  the 
introduction  of  the  bacterium  is  followed  by  the  production  of  a 
local  lesion.  This  always  indicates  some  degree  of  local  immunity, 
and  may  be  regarded  as  an  attempt  to  localize  the  organism  and 
prevent  its  further  spread.  And  the  nature  and  severity  of  the 
local  lesion  stand  in  close  relation  to  the  severity  of  the  infection 

3 


34  THE    LOCAL    LESION 

and  the  degree  of  the  immunity.  For  example,  in  severe  and 
rapidly  fatal  infections  from  post-mortem  wounds — i.e.,  where  the 
infection  is  virulent  and  the  immunity  but  slight — there  is  very 
little  local  reaction  and  very  little  glandular  enlargement,  the 
process  being  septicaemic  from  the  first.  Where  the  infective  and 
protective  forces  are  equally  matched  the  local  lesions  are  more 
developed;  inflammation,  and  usually  suppuration,  occur  at  the 
site  of  the  wound,  and  the  glands  enlarge  and  may  suppurate ; 
and  when  the  infection  is  so  feeble  as  to  be  quite  unable  to  cope 
with  the  immunity,  the  local  lesion  is  the  sole  result  of  the  infec- 
tion. Eyre  gives  a  similar  example  in  the  results  of  injecting 
similar  doses  of  pneumococci  into  rabbits  of  different  ages.  The 
young  animal  is  most  susceptible,  and  in  it  death  occurs  within 
forty-eight  hours  from  septicaemia,  and  there  is  but  little  local 
reaction.  In  half-grown  animals  the  local  lesion  is  more  developed, 
and  is  gelatinous  or  fibrinous,  containing  many  leucocytes,  and 
the  animal  lives  several  days.  In  old  rabbits  quite  definite  pus  is 
formed,  and  the  animal  lives  longer,  and  may  recover  completely. 
Hence  suppuration  may  be  regarded  as  a  proof  that  the  defensive 
and  infecting  forces  are  fairly  balanced,  and  that  either  may  be 
victorious  in  the  conflict. 

The  other  local  lesions  need  not  be  discussed  at  length,  but  the 
case  of  tubercle  and  the  allied  diseases  requires  a  brief  notice. 
Here  the  lesion  indicates  the  presence  of  a  very  considerable 
degree  of  immunity  to  the  toxin,  for  the  structure  of  a  tubercle  is 
exactly  similar  to  that  of  the  cellular  reaction  to  many  feebly 
irritating  foreign  bodies — e.g.,  unabsorbable  ligatures,  substances 
from  which  it  is  clear  no  potent  toxin  can  be  given  off;  but  it 
also  indicates  that  there  is  a  defect  in  the  mechanism  by  which 
the  bacilli  should  be  removed,  since  the  process  is  (for  a  time  at 
least)  a  progressive  one.  Here  the  walling-in  of  the  infected  area 
which  occurs  as  the  result  of  the  reaction  of  the  tissues  may  be 
taken  to  be  a  defensive  process,  but,  as  we  shall  have  occasion  to 
see,  it  is  one  of  doubtful  utility. 

EARLY  THEORIES  OF  IMMUNITY. — Before  turning  to  the  dis- 
cussion of  the  nature  of  immunity  in  the  light  of  our  present 
knowledge,  it  will  be  convenient  to  insert  a  short  account  of  some 
of  the  early  theories  of  the  subject,  which  are  in  the  main  of 
historic  interest  only.  They  have  served  their  purpose  as  a  point 
of  departure  for  subsequent  research. 

Of  such  nature  was  Pasteur's  theory  of  exhaustion,  the  earliest 


INTRODUCTORY  AND   GENERAL  35 

attempt  at  a  scientific  explanation  of  the  facts  of  recovery  from, 
and  subsequent  immunity  to,  the  infectious  diseases.  Pasteur  was 
a  chemist,  and  was  only  led  to  the  study  of  bacteriology  by  the 
pursuit  of  chemical  investigations  into  examining  reactions  which 
he  proved  to  be  due  to  micro-organisms.  His  theory  was  a 
chemical  one.  A  certain  amount  of  food  is  necessary  for  each 
bacterium,  and  when  the  total  amount  contained  in  a  given  solu- 
tion is  used  up  the  growth  of  the  bacteria  must  cease.  For 
example,  if  we  take  a  dilute  solution  of  sugar  (containing  the 
necessary  salts,  etc.),  and  inoculate  it  with  yeast,  the  cells  will 
begin  to  divide  and  multiply  with  great  rapidity.  After  a  time 
the  growth  ceases,  and  it  will  not  be  resumed  if  we  inoculate  the 
fluid  with  an  additional  amount  of  yeast.  We  may  compare  the 
test-tube  to  the  patient,  the  yeast  to  the  pathogenic  organism,  and 
the  process  of  fermentation  to  the  disease,  and  we  may  say  that 
the  fluid  has  recovered  from  the  disease  and  is  now  immune  to  it. 
This  immunity  depends  upon  the  absence  of  sugar,  which  was 
used  up  by  the  yeast  cells,  and  if  more  sugar  be  added  the  process 
of  fermentation  may  be  restarted  by  a  fresh  inoculation,  or  by  the 
yeast  still  remaining. 

The  theory  can  easily  be  disproved,  from  the  fact  that  bacteria 
may  grow  well  enough  in  the  dead  tissues  and  fluids  of  immune 
animals ;  and,  secondly,  because  immunity,  as  we  have  seen,  may 
be  produced  (in  some  cases)  by  the  injection  of  the  chemical  pro- 
ducts of  the  bacteria,  substances  which  can  hardly  use  up  food 
materials.  The  theory  has,  however,  been  recently  revived  in  a 
modified  form  by  Ehrlich,  who  considers  that  there  is  sufficient 
evidence  for  the  occurrence  of  this  form  of  immunity  in  certain 
cases.  He  calls  it  atreptic  immunity. 

The  retention  hypothesis  of  Chauveau  is  the  exact  opposite  of 
Pasteur's.  Several  observers  showed  that  the  growth  of  micro- 
organisms in  fluid  media  might  cease  spontaneously  whilst 
abundant  food  material  remained  unutilized.  This  was  found  to 
be  due  to  the  presence  of  certain  products  of  metabolism,  which, 
like  carbon  dioxide  in  the  case  of  animals,  act  as  poisons  to  the 
organism  which  produces  them.  For  instance,  the  fermentation 
of  sugar  by  yeast  is  found  to  cease  when  about  14  per  cent,  of 
alcohol  is  present,  and  if  a  strong  solution  be  taken  the  process 
will  stop  at  this  point,  but  can  be  started  again  if  the  alcohol  be 
removed  by  distillation.  Here  the  fermentation  is  stopped  by 
alcohol,  a  product  of  metabolism  of  the  yeast  cell,  which  acts  as  a 

3—2 


36  THEORY    OF    RETENTION 

poison  on  the  organism  producing  it.  The  theory  of  immunity 
based  on  these  facts  is  obvious.  Bacteria  growing  in  the  body 
will  yield  substances  inimical  to  the  continued  growth  of  the 
organism,  so  that  they  will  die  out  and  recovery  ensue,  and  the 
body  will  remain  immune  as  long  as  these  substances  are  retained 
therein.  This  theory  accounts  well  for  the  production  of  immunity 
by  injections  of  the  toxins  and  other  soluble  products  of  bacteria. 
It  is  negatived  by  the  fact  that  bacteria  may  grow  in  the  blood  and 
tissues  of  immune  animals,  and  is  improbable  if  we  consider  that 
immunity  may  last  for  many  years,  and  that  it  is  extremely 
improbable  that  substances  (necessarily  soluble)  should  be  retained 
in  the  body  for  so  long  a  time. 

We  shall  now  proceed  to  a  study  of  the  more  modern  views, 
and  in  doing  so  it  will  be  convenient  to  deal  with  the  subjects  of 
immunity  to  toxins  and  immunity  to  bacteria  in  separate  sections. 
Of  course,  in  most  cases  they  run  parallel  to  one  another:  an 
animal  contracts  a  disease  because  its  fluids  and  tissues  cannot 
kill  the  pathogenic  bacteria  offhand,  and  because  its  cells  are  sus- 
ceptible to  the  action  of  the  toxin,  and  vice  versa.  This  is  not 
necessarily  the  case,  however,  and  the  two  phenomena  may  be 
entirely  independent. 

The  subject  of  immunity  of  toxins  is  on  the  whole  the  more 
important  of  the  two,  the  simpler  (though  complex  enough),  and 
the  best  understood.  It  will  be  best  to  deal  with  it  first. 


CHAPTER  II 
ON  THE  NATURE  OF  TOXINS 

THE  fact  that  the  pathogenic  action  of  any  organism  is  dependent 
entirely,  or  almost  entirely,  on  that  of  the  ^toxins  which  it  pro- 
duces renders  it  necessary  to  make  a  brief  study  of  these 
substances  before  considering  the  method  in  which  the  infected 
animal  reacts  to  the  organism,  and  defends  itself  against  infection. 
In  doing  so  we  must  distinguish  clearly  between  the  specific 
toxins  which  are  produced  by  any  organism  and  the  non-specific 
and  less  important  poisons  which  it  may  also  elaborate.  The 
difference  is  a  fundamental  one.  Numerous  bacteria  produce 
by-products  of  metabolism,  excreta,  etc.,  which  are  comparatively 
simple  chemical  substances  of  definite  composition  ;  for  example, 
acids,  alkalis,  alcohol,  ptomains,  nitrites,  etc.  These  may  be 
poisonous,  and  may,  in  some  cases  at  least,  play  a  part  of  some 
importance  in  the  production  of  the  symptoms  of  the  disease. 
The  cholera  vibrio,  for  instance,  produces  nitrites  in  considerable 
amount,  and  since  the  symptoms  of  cholera  have  some  resem- 
blance to  those  of  nitrite  poisoning,  it  is  conceivable  that  those 
substances  may  be,  to  some  extent  at  least,  the  active  causes  of 
the  disease,  and  these  nitrites  might  be  regarded  as  the  toxins  of 
the  cholera  vibrio.  This,  however,  is  not  the  case,  and  the  true 
toxins  are  quite  different  in  nature,  as  is  shown  by  many  facts, 
especially  by  the  proof  that  cholera  vibrios  which  have  no  longer 
the  power  of  producing  nitrites  may  still  cause  infection  in 
susceptible  animals. 

The  specific  bacterial  toxins  differ  from  these  poisonous  sub- 
stances in  many  important  particulars.  They  are,  as  a  rule, 
formed  only  in  very  small  amounts,  and  are  extremely  powerful. 
For  example,  the  toxin  of  tetanus  may  readily  be  obtained  in  so 
poisonous  a  solution  that  ToV TF  c-c-  w*ll  kill  a  guinea-pig  in  a  day 
or  two,  and  of  this  solution  only  a  very  small  fraction  even  of 
the  dried  residue  consists  of  toxin.  They  are  not  simple  chemical 

37 


3  TOXINS — THEIR   FRAGILITY 

substances,  and  their  exact  nature  is  as  yet  unknown.  This  may 
be  due  in  part  to  the  minute  amounts  which  are  formed,  and  in 
part  to  the  difficulties  which  prevent  their  being  obtained  in  a 
pure  state  ;  but  there  are  other  reasons,  to  which  we  shall  revert 
later,  for  this  complexity.  Further,  they  are,  with  a  few  ex- 
ceptions, very  fragile  substances,  and  are  readily  destroyed  by 
the  action  of  many  agents,  and  especially  by  heat.  Nearly  all 
the  bacterial  toxins  are  rendered  inert  by  boiling,  and  many  of 
them  by  a  short  exposure  to  a  temperature  of  60°  or  70°  C. 
They  are  usually  destroyed  by  gastric  digestion,  so  that  they  are 
without  action  when  administered  by  the  mouth. 

A  considerable  amount  of  attention  has  been  paid  to  this 
question,  since  it  would  be  desirable,  if  possible,  to  replace  hypo- 
dermic injections  of  vaccines,  etc.,  by  oral  or  rectal  administration. 
In  general  terms  the  statement  made  above  holds  good  :  toxins 
administered  by  the  mouth  are  not  absorbed  as  such,  and  do  not 
produce  the  characteristic  symptoms  of  the  disease.  In  some 
cases,  however,  there  is  reason  to  believe  that  a  small  amount 
of  absorption,  probably  of  the  toxin  in  an  altered  form,  does 
occur,  and  a  certain  degree  of  immunity  may  be  produced  by 
the  oral  administration  of  killed  cultures  of  typhoid  bacilli,  and 
possibly  of  tubercle  bacilli.  But  this  method  has  only  one  advan- 
tage— its  painlessness — over  the  hypodermic  method,  whereas  its 
uncertainty  renders  it  extremely  undesirable.  There  can  be  no 
doubt  that  the  advantage  of  giving  an  exactly  measured  dose,  with 
the  certainty  that  every  particle  will  be  absorbed  and  act  in  the 
way  desired,  will,  under  ordinary  circumstances,  render  the  hypo- 
dermic method  infinitely  preferable.  To  administer  infinitesimal 
doses  of  killed  tubercle  bacilli  or  of  TR  to  an  infant  who  may  be 
swallowing  large  doses  of  living  and  dead  bacilli  in  milk,  sputum, 
etc.,  does  not  appear  rational,  and  the  clinical  evidence  in  its  favour 
is  entirely  unconclusive.  In  the  case  of  ricin,  about  a  hundredth 
part  of  the  toxin  given  by  the  mouth  is  absorbed  as  such — i.e.t  the 
minimal  lethal  dose  on  oral  administration  is  about  100  times 
as  large  as  the  lethal  dose  of  the  same  preparation  given  sub- 
cutaneously  (Stillmarck).  Ricin  is,  however,  far  more  resistant  to 
the  action  of  digestive  enzymes  than  are  the  exotoxins. 

The  most  important  feature  of  the  bacterial  toxins  is  their 
relation  to  immunity.  It  is  possible  in  all  cases  to  render  a 
susceptible  animal  immune  to  their  action  by  the  injection  of  the 
toxins  in  suitable  doses  at  suitable  intervals,  though  in  some 


ON   THE    NATURE   OF  TOXINS  39 

cases  the  task  is  a  difficult  one.  This  is  not  the  case  with  the 
non-specific  toxins.  It  is  true  that  in  a  few  isolated  instances  we 
are  able  to  increase  slightly  the  resistance  of  an  animal  to  the 
simple  chemical  poisons  (e.g.,  to  alkaloids  such  as  morphine),  but 
these  apparent  exceptions  hardly  interfere  with  the  utility  of  the 
general  rule.  Further,  and  more  important,  an  animal  immunized 
to  the  action  of  a  toxin  is  also  protected  against  the  pathogenic  action 
of  the  bacterium  which  produces  it,  and  vice  versa.  Thus  an  animal 
which  has  been  rendered  immune  to  the  toxin  of  tetanus  by  re- 
peated injections  of  that  substance  is  also  immune  to  infection 
with  the  living  cultures  of  the  bacillus,  and  an  animal  which  has 
successfully  survived  an  infection  with  the  tetanus  bacillus  is 
thereby  rendered  in  some  degree  immune  to  the  action  of  tetanus 
toxin.  This  method  of  immunization  with  the  bacterial  toxins 
(the  so-called  "  chemical  vaccination  ")  is  of  the  utmost  impor- 
tance in  practice.  It  was  introduced  by  Smith  and  Salmon,  who 
showed  that  it  was  possible  to  immunize  pigeons  against  living 
cultures  of  the  hog-cholera  bacillus  by  means  of  the  sterilized 
products  of  that  organism. 

When  this  method  is  applicable  it  supplies  us  with  a  test  as  to 
the  specificity  of  a  toxic  substance  which  we  have  isolated  from  a 
culture  of  a  bacterium,  or  from  the  organs  of  an  animal  which 
has  been  killed  by  an  infection.  The  substance  must  be  poisonous 
for  animals  which  are  susceptible  to  the  infection  in  question,  and 
it  must  be  harmless  to  animals  which  have  been  immunized  to 
the  organism  ;  on  the  other  hand,  it  must  immunize  animals  both 
to  its  own  action  and  to  that  of  the  bacterium  when  injected  in 
a  living  state.  These  conditions  are  never  fulfilled  by  the  non- 
specific toxins. 

There  are  a  few  apparent  exceptions  to  this  rule,  but  they  fail 
to  stand  investigation,  being  based  on  the  fact  that  it  is  easier  to 
render  an  animal  refractory  to  a  living  organism  than  to  its  toxin. 
Thus  an  animal  which  has  been  injected  with  the  filtered  products 
of  certain  organisms  may  be  rendered  immune  to  infection  with 
those  organisms,  but  remain  as  susceptible  as  before  to  their 
toxins.  But  this  is  due  to  the  fact  that  the  animal  has  been 
immunized  but  partially ;  if  the  process  be  carried  further  the 
animal  will  be  rendered  refractory  to  both. 

Again,  an  animal  which  has  been  immunized  to  the  toxin  of 
one  bacterium  remains  as  susceptible  as  before  to  the  action  of 
another  toxin  or  bacterium.  A  horse  which  has  been  immunized 


40  THE  EXOTOXINS 

to  diphtheria  toxin  (e.g.,  in  the  production  of  diphtheria  antitoxin) 
will  be  just  as  susceptible  to  tetanus  toxin  as  a  normal  animal. 
In  a  very  few  cases  the  law  does  not  hold.  The  only  well- 
authenticated  example  of  this  sort  is  the  antagonism  which 
animals  display  to  anthrax  after  injection  with  the  products  of 
B.  pyocyaneus. 

These  preliminary  considerations  will  serve  to  show  the  more 
important  criteria  by  which  the  nature  of  a  bacterial  product  may 
be  determined,  and  its  nature  as  a  true  toxin  established. 

These  toxins  were  soon  found  to  fall  into  two  main  groups — the 
extracellular  or  soluble  toxins,  or,  as  we  shall  call  them,  the  exo- 
toxins,  and  the  intracellular  insoluble  toxins,  or  endotoxins.  We 
shall  consider  these  substances  in  turn. 

THE  EXOTOXINS. 

The  exotoxim  are  substances  which  are  given  off  in  a  free  state 
when  the  bacteria  are  grown  in  a  suitable  culture  medium  outside 
the  body,  and  can  usually  be  separated  by  simple  filtration  (through 
a  Pasteur  or  Berkefeld  filter)  from  the  organisms  which  produce 
them.  We  may  consider  them  provisionally  as  the  specific  secre- 
tions or  excretions  of  the  bacteria.  They  are  not  formed  by  all 
pathogenic  bacteria — that  is,  in  the  present  state  of  bacteriological 
science  no  suitable  culture  media  have  been  found  in  which  certain 
organisms  will  produce  a  soluble  toxin.  The  three  most  impor- 
tant organisms  which  do  so  are  the  B.  tetani,  B.  diphtheria, 
and  the  B.  botulismus.  These  toxins,  the  first  two  especially, 
are  substances  of  the  greatest  interest,  since  they  have  been 
submitted  to  a  most  profound  examination,  and  our  knowledge 
of  the  structure  of  bacterial  toxins,  of  their  action  on  the  body, 
and  of  the  production  of  immunity  thereto,  is  based  almost  entirely 
on  the  results  thus  obtained.  In  addition  to  these,  there  are  sub- 
stances which  are  much  less  toxic — if,  indeed,  toxic  at  all — and 
which  fail  to  fulfil  our  definitions  of  a  specific  toxin,  since  an 
animal  which  has  been  immunized  thereto  is  not  necessarily 
immune  to  the  organism,  but  which  have  many  points  in  common 
with  the  true  toxins,  and  will  be  considered  in  this  connection. 
These  are  the  bacterial  cytolysins  and  hsemolysins, T  substances 

1  Haemolysis,  or  the  liberation  of  haemoglobin  from  red  blood-corpuscles, 
may  be  brought  about  by  a  variety  of  agents,  which  fall  under  three  main 
headings :  (i)  Simple  chemical  substances,  such  as  distilled  water,  ether, 
acids,  etc.,  which  act  osmotically,  or  by  a  direct  solution  of  the  strorna  of 


ON    THE    NATURE   OF   TOXINS  41 

which  have  the  power  of  dissolving  living  cells  or  red  blood- 
corpuscles  respectively  from  susceptible  animals. 

In  dealing  with  these  substances  we  will  consider  firstly  their 
action,  secondly  their  structure,  and  thirdly  what  has  been  estab- 
lished concerning  their  chemical  relationships  with  other  sub- 
stances. The  last  is  comparatively  unimportant. 

i.  Action  of  Toxins. — The  results  of  the  injection  of  a  toxin 
into  a  living  and  susceptible  animal  depend,  in  most  instances, 
on  the  dose  injected.  If,  for  instance,  we  inject  a  large  amount  of 
the  filtered  broth  in  which  the  tetanus  bacillus  has  been  growing 
for  a  month  or  so,  and  which  in  consequence  contains  tetanus 
toxin,  the  animal  (a  guinea-pig,  for  example)  will  develop  the 
rigidities,  spasmodic  contractions  of  the  muscles,  etc.,  charac- 
teristic of  tetanus ;  and  these  make  their  appearance  after  an 
interval  of  some  hours,  during  which  period  the  animal  shows 
no  symptoms  whatever  of  the  disease.  Great  stress  was  laid 
at  one  time  on  the  occurrence  of  this  "  latent  period,"  since  it  was 
thought  to  be  peculiar  to  the  bacterial  toxins  (and  to  the  similar 
substances  of  animal  and  vegetable  origin),  and  to  distinguish 
them  sharply  from  other  poisons,  alkaloids,  etc.  This  is  hardly 
correct.  It  is  true  that  in  most  cases  of  intoxication  by  bacterial 
toxins  there  is  a  latent  period,  but  in  a  few  it  is  practically  absent 
The  most  interesting  example  is  the  "  Nasik  "  vibrio,  an  organism 
allied  to  that  of  cholera.  This  produces  an  exotoxin  (though  not 
a  very  powerful  one  in  the  sense  that  it  kills  in  small  doses), 
which  proves  fatal  on  intravenous  injection  into  a  rabbit  after  a 
period  of  ten  to  thirty  minutes,  and  symptoms  are  produced  before 
this.  On  the  other  hand,  some  of  the  alkaloids,  and  notably 
colchicine,  display  a  well-marked  latent  period.  The  phenomenon, 
therefore,  is  not  absolutely  peculiar  to,  nor  characteristic  of,  the 
toxins ;  but  since  it  is  so  commonly  displayed  by  them,  it  calls  for 
some  investigation.  Moreover,  we  must  assume  that  part  at 
least  of  the  incubation  period  of  an  infective  disease  is  taken  up 
by  the  latent  period  of  the  bacterial  toxin,  a  circumstance  which 
invests  it  with  especial  interest.  Thus  a  horse  which  Madsen 

the  corpuscles  or  of  parts  thereof;  (2)  the  simple  organic  haemolysins,  which 
include  the  bacterial  haemolysins  dealt  with  above,  the  haemolysins  of  vegetable 
origin  (such  as  ricin,  etc.),  and  some  of  the  haemolysins  of  animal  origin  ; 
and  (3)  the  compound  haemolysins,  all  of  animal  origin,  which  will  be  dealt 
with  subsequently.  These  groups  differ  profoundly  in  their  action,  and  must 
be  kept  quite  distinct. 


42  ACTIONS    OF   TOXINS 

was  treating  for  the  production  of  diphtheria  antitoxin  developed 
tetanus,  and  tetanus  toxin  was  found  in  a  sample  of  blood  collected 
five  days  before  the  development  of  symptoms. 

On  diminishing  the  amount  of  the  toxin  which  we  inject,  we 
find  that  the  latent  period  becomes  gradually  longer,  and  the 
duration  of  the  disease  (i.e.,  the  time  between  the  first  develop- 
ment of  symptoms  of  intoxication  and  the  fatal  issue)  also  lengthens. 
By  diminishing  the  dose  gradually  we  can  find  an  amount  which 
will  just  kill  the  animal  in  question  in  a  given  number  of  days, 
and,  provided  the  test  animals  used  are  approximately  the  same 
in  age  and  weight,  we  shall  find  that  this  amount,  the  "  minimal 
lethal  dose,"  is  fairly  constant  for  animals  of  the  same  species. 
Thus,  in  the  standardization  of  diphtheria  antitoxin  the  first  step 
is  the  estimation  of  the  minimal  lethal  dose  of  the  toxin,  and  for 
this  purpose  it  is  customary  to  use  guinea-pigs  weighing  from 
250  to  280  grammes,  and  to  fix  a  time-limit  of  four  days.  It  is 
found  that  the  minimum  lethal  dose  is  the  same,  within  close 
limits,  for  all  test  animals,  and  that  if  a  series  similar  in  size  and 
weight  be  inoculated  with  the  same  dose,  the  majority  will  die 
within  a  few  hours  of  one  another.  This  fact  enables  diphtheria 
antitoxin  to  be  titrated  with  some  approach  to  chemical  accuracy, 
the  test  guinea-pig  being  used  as  the  indicator. 

On  reducing  the  dose  still  further,  we  find  that  the  incubation 
period  is  still  further  prolonged,  that  the  symptoms  are  less  severe, 
and  that  death  may  not  take  place,  or  only  do  so  at  a  later  period 
than  that  which  has  been  fixed  for  the  minimal  lethal  dose.  Thus, 
in  antitoxin-testing  a  dose  of  toxin  which  does  not  kill  in  five  days 
is  regarded  as  a  sublethal  dose,  although  death  may  take  place  at 
a  later  date — perhaps  much  later. 

On  giving  still  smaller  doses  the  symptoms  take  still  longer  to 
develop,  are  still  slighter,  and  are  followed  by  recovery,  and  the 
animal  may  then  present  a  certain  degree  of  immunity  to  the  toxin 
and  to  the  organism  producing  it.  On  the  other  hand,  under 
certain  circumstances  it  may  be  more  than  usually  sensitive  to 
the  action  of  the  toxin  in  question. 

These  phenomena  present  some  points  of  comparison  with 
those  which  are  presented  in  the  action  of  the  soluble  enzymes, 
such  as  pepsin.  In  each  case  an  excessively  minute  amount  of 
the  active  substance  will  produce  the  given  effect,  and  in  each 
the  effect  is  more  rapid  if  a  larger  amount  be  used.  In  either 
case  there  is  a  latent  period  of  longer  or  shorter  duration  before 


ON    THE    NATURE    OF   TOXINS  43 

the  peculiar  chemical  action  is  manifested.     There  are  several 
other  analogies  between  the  soluble  enzymes  and  the  exotoxins. 

(a)  The  soluble  enzymes  are,  without  exception,  all  produced 
by  living  animal  or  vegetable  cells,  and  are  either  secreted  or 
excreted  by  them,  or  remain  locked  in  their  protoplasm.     The 
bacterial  toxins,  in  the  same  way,  are  all  formed  and  eliminated 
by  living  bacteria ;  or,  in  the  case  of  the  endotoxins,  retained 
in   the   cell.     In  other   words,   both    extracellular   enzymes  and 
exotoxins  are  products  of  metabolism  given  off  during  the  life  of 
a  living  organism.     Further,  both  substances  represent  a  method 
in  which  the  organism  attempts  to  modify  its  environment  and 
render  it  more  suitable  :  the  animal  secretes  pepsin  into  its  stomach 
in  order  to  modify  the  ingested  proteids  and  render  them  suitable 
for  food,  and  the  tetanus  bacillus  produces  toxin  in  a  living  animal 
because  it  is  in  itself  but  little  adapted  to  grow  in  living  tissues, 
but  can  do  so  easily  when  these  tissues  have  been  injured  by  the 
action  of  toxin.     The  spores  of  tetanus  which  have  been  washed 
free  from  all  traces  of  toxin  have  no  power  of  producing  tetanus 
when  injected  into  an  animal,  and  are  rapidly  taken  up  by  the 
leucocytes,  or  otherwise  dealt  with  by  the  tissues ;  but  if  a  minute 
amount  of  toxin  be  injected  at  the  same  time  the  bacteria  can 
resist  the  leucocytes  and  tissues,  which  are  injured  thereby,  and 
continue  to  grow  and  produce  fresh  toxin,  giving  rise  to  fatal 
tetanus. 

(b)  It  is  capable  of  proof  that  enzymes  commence  their  action 
on  the  substances  which  they  attack  by  forming  a  combination 
therewith.     Thus  the  first  effect  of  the  addition  of  pepsin  to  fibrin 
is  the  formation  of  a  compound  between  the  two  substances,  as 
shown  by  the  fact  that,  if  the  fibrin  be  thoroughly  washed  at 
a   temperature   near   the   freezing-point  until  all  traces  of  free 
enzyme  are  washed  away,  it  will  still  undergo  digestion  when 
raised  to  the  body  temperature.     Further,  the  enzyme  is  less 
easily  destroyed  by  heat  when  it  has  combined  with  the  fibrin. 

In  a  similar  way  it  is  capable  of  proof  that  the  toxins  unite 
chemically  with  the  cells  of  susceptible  animals.  The  proof  may 
be  deduced  from  the  fact  that  if  toxin  be  injected  intravenously 
into  a  susceptible  animal  it  rapidly  disappears  from  the  blood, 
although  it  does  not  escape,  or  only  to  a  very  small  extent,  in  the 
secretions.  When  the  injection  is  made  into  insusceptible  animals 
it  may  disappear  by  a  process  to  be  discussed  subsequently,  or 
may  persist  for  long  periods.  Thus  in  one  case  Metchnikoff 


44  COMBINATION   OF   TOXINS   AND   TISSUES 

was  able  to  demonstrate  the  presence  of  tetanus  toxin  in  the 
tortoise,  which  is  insusceptible  to  the  action  of  that  substance, 
at  a  period  of  four  months  after  the  injection.  That  the  disap- 
pearance which  occurs  in  susceptible  animals  is  actually  due  to 
a  combination  of  the  toxin  with  the  tissues  of  the  body,  and  not 
to  its  destruction  or  elimination,  is  shown  by  the  fact  that  the 
tissus  of  an  animal  which  has  been  injected  with  tetanus  toxin, 
but  which  no  longer  contains  that  substance  in  the  blood,  may 
produce  tetanus  when  injected  into  a  susceptible  animal.  In  the 
case  of  fowls  it  seems  that  this  power  of  combining  with  tetanus 
toxin  is  most  marked  in  the  leucocytes.  Again,  it  is  possible 
to  reproduce  the  absorption  of  tetanus  toxin  by  fresh  tissues  in 
vitro.  This  has  been  especially  studied  by  Ignowtowsky,  who 
showed  that  emulsions  of  liver,  kidney,  spleen,  etc.,  have  the 
power  to  absorb  tetanus  toxin,  but  that  the  subsequent  injection 
of  these  cells  will  produce  the  symptoms  of  the  disease. 

It  ought  to  follow  logically  that  the  toxin  will  combine  especially 
with  those  cells  and  tissues  which  are  acted  upon  by  it  in  the 
living  body,  and  in  all  probability  this  is  the  case.  The  proof, 
however,  is  somewhat  difficult.  Wassermann  apparently  proved 
the  point  by  his  demonstration  of  the  fact  that  tetanus  toxin  is 
absorbed  and  neutralized  by  an  emulsion  of  the  central  nervous 
system,  and  not  by  that  of  any  other  organ,  although,  as  has 
been  mentioned  above,  it  is  absorbed  by  other  tissues.  Now, 
tetanus  toxin  acts  entirely,  or  almost  entirely,  on  the  central 
nervous  system,  and  this  well-known  and  oft-quoted  experiment 
appears  to  constitute  a  proof  of  the  point  at  issue.  The  exact 
interpretation  of  Wassermann's  experiment  appears,  however,  to 
be  doubtful,  and  it  is  hardly  safe  to  rely  on  it  as  a  proof  of  the 
point. 

With  the  bacterial  haemolysins,  which,  although  of  feeble 
toxicity,  are  in  every  other  respect  identical  with  the  exotoxins, 
we  are  on  surer  ground.  A  filtered  broth  culture  of  the  tetanus 
bacillus  contains  the  specific  toxin  (tetanospasmin),  and  in  addi- 
tion a  second  substance,  which  has  the  power  of  dissolving  red 
blood-corpuscles  when  kept  at  a  temperature  near  that  of  the 
body.  At  a  low  temperature  they  do  not  act  in  this  way  ;  but  if 
red  corpuscles  be  added  in  suitable  amount  to  a  solution  of  tetano- 
lysin  at  a  temperature  of  o°  C.  and  centrifugalized,  the  supernatant 
fluid  has  no  longer  the  power  of  producing  haemolysis.  On  the 
other  hand,  the  red  corpuscles,  even  after  washing  with  normal 


ON   THE    NATURE   OF   TOXINS  45 

saline  solution  to  remove  all  traces  of  free  haemolysin,  are  dissolved 
when  raised  to  the  body  temperature.  In  other  words,  the 
specific  haemolysin  of  tetanus  can  form  a  combination  with  the 
structures  on  which  they  act.  Numerous  similar  examples  will 
be  met  with. 

(c)  In  some  of  the  specific  exotoxins,  notably  that  of  tetanus,  we 
meet  with  a  similar  dependence  on  a  suitable  temperature  for  the 
development  of  their  toxic  action,  a   property  in  which  again 
they  resemble  the  soluble  enzymes.     The  most  striking  example 
is  obtained  by  a  study  of  the  action  of  tetanus  toxin  on  the  frog, 
which,  in  common  with  all  cold-blooded  animals,  is  but  slightly 
susceptible  to  its  action.     If,  however,  the  frogs  be  kept  in  an 
elevated  temperature — 30°  C.  or  higher — they  develop  the  typical 
symptoms  of  the  disease  after  five  days  or  thereabouts.     Now 
Morgenroth   has  shown  that  the  toxin   unites  with  the  central 
nervous  system  at  a  low  temperature  (8°  C.),  but  without  the  de- 
velopment of  symptoms.    For  the  production  of  these  a  high  tem- 
perature is  necessary,  exactly  as  in  the  case  of  the  combination 
of  tetanolysin  with  red  blood-corpuscles  and  the  solution  of  the 
latter. 

(d)  These  and  similar  researches  lead  us  to  distinguish  between 
two  faculties  of  a  toxin — that  of  combining  and  that  of  injuring ; 
and  the  fact  that  in  some  instances  these  processes  can  take  place 
at  different  temperatures  leads  us  to  the  belief  that  they  are  quite 
different  properties  of  the  toxin.     In  other  words,  the  mere  union 
of  a  toxin  with  a  cell  is  not  sufficient  to  cause  injury  to  the  latter. 
This  is  susceptible  of  proof.    In  Ehrlich's  elaborate  studies  on  the 
standardization  of  diphtheria  antitoxin  he  first  obtained  a  speci- 
men of  diphtheria  antitoxin,  and  determined  its  minimum  lethal 
dose  for  test  guinea-pigs.     For  the  sake  of  simplicity  we  will 
suppose  that  for  a  given  sample  of  toxin  this  was  T^  c.c. — i.e.9 
that  amount  of  the  filtered  broth  culture  of  the  diphtheria  bacillus 
would  just  kill  a  guinea-pig  weighing  250  grammes  in  four  days. 
Further,  let  us  suppose  that  we  have  a  standard  sample  of  anti- 
toxin of  which  i  c.c.  just  neutralizes  i  c.c.  of  toxin  (100  lethal 
doses),  so  that  the  mixture  of  the  two  causes  no  symptoms  when 
injected  into  a  test  animal.     Diphtheria  antitoxin  is  a  relatively 
stable  substance,  and  can  be  preserved  in  a  dry  state,  at  a  low 
temperature,  for  long  periods  if  light  and  air  are  excluded.     It  is 
thus  possible  to  re-test  the  sample  of  toxin  with  a  precisely  similar 
solution  of  antitoxin  after  some  months.     When  this  is  done,  it  is 


46  TOXINS   AND   TOXOIDS 

found  that  it  will  have  fallen  off  in  potency ;  for  example,  it  may 
take  gL  c.c.  to  kill  a  guinea-pig.  It  might  be  supposed  that  this 
was  due  to  a  complete  destruction  of  half  the  toxin,  but  this  is  not 
the  case.  If  it  were  so,  we  should  find  that  to  neutralize  i  c.c. 
(  =  50  lethal  doses)  we  should  require  \  c.c.  of  antitoxin,  since  the 
latter  has  not  altered  in  potency.  As  a  matter  of  fact,  we  find 
that  we  still  require  i  c.c.  of  antitoxin  ;  in  other  words,  the 
diminution  of  the  toxic  power  of  the  solution  has  not  been  accom- 
panied by  a  diminution  in  its  combining  capacity  for  antitoxin. 
The  explanation  given  by  Ehrlich,  and  fully  proved  by  analogy 
with  numerous  other  similar  phenomena,  is  that  part  of  the  toxin 
has  altered  into  a  substance  which  retains  its  power  of  uniting 
with  antitoxin  (and,  as  we  shall  show  later,  with  the  tissue  cells), 
but  which  has  been  deprived  of  its  toxicity.  Toxin  which  has 
undergone  this  change  is  called  toxoid.  Haemolysin  also  appears 
to  undergo  a  similar  change  into  haemolysoid,  and  the  rapid  loss  of 


.    #< 

I  J 


FIG.  5.— A  MOLECULE  OF  TOXIN  WITH  ITS  HAPTOPHORE  (a)  AND 
TOXOPHORE  (6)  GROUPS. 

On  the  right  a  similar  molecule,  which  has  lost  its  toxophore  group,  and 
become  converted  into  toxoid. 

activity  which  tetanolysin  undergoes  is  very  probably  due  to  a 
change  into  that  substance. 

The  alteration  of  the  toxin  to  toxoid  can  be  best  explained 
by  supposing  that  the  power  of  entering  into  combination  and  the 
power  of  intoxication  reside  in  two  different  parts — which  we  may 
regard  as  groups  of  atoms — of  the  molecule  of  toxin,  and  by 
further  supposing  that  the  combining  group  is  a  relatively  stable 
one,  and  that  the  toxic  group  is  easily  destroyed.  In  the  very 
convenient  nomenclature  introduced  by  Ehrlich,  and  now  uni- 
versally adopted,  the  group  of  atoms  which  has  the  power  of 
entering  into  chemical  combination  with  the  living  protoplas 
or  with  antitoxin  is  called  the  haptophore  grotip,  whilst  the  portion 
on  which  the  toxic  action  depends  is  called  the  toxophore  group. 
The  change  of  toxin  into  toxoid,  or  of  haemolysin  into  haemolysoid, 
consists  in  a  destruction  of  the  toxophore  group,  with  retention  of 
the  more  stable  haptophore  group  (Fig.  5.)  From  what  has  been 
said  as  to  the  dependence  of  the  phenomena  of  intoxication  on  a 


ON   THE    NATURE   OF   TOXINS  47 

temperature  approaching  that  of  the  body,  it  follows  that  the  hapto- 
phore  group  can  functionate  at  a  low  temperature  (o°  to  10°  C.), 
while  the  toxophore  group  can  only  do  so  at  a  fairly  high  one. 
Looked  at  in  this  way,  the  process  of  intoxication  with  an  endo- 
toxin,  or  of  haemolysis  with  a  bacterial  haemolysin,  may  be  divided 
into  two  stages :  in  the  first  place,  the  haptophore  group  of  the 
toxin  or  haemolysin  combines  with  the  protoplasm  or  with  the 
stroma  of  the  red  corpuscle,  and  in  the  second  the  toxophore 
group  exerts  its  action,  and  the  cell  is  poisoned  or  the  red  corpuscle 
dissolved.  The  phenomena  of  tetanus  in  frogs  is  thus  readily 
explicable. 

We  shall  see  several  other  examples  of  substances  in  which  it 
is  possible  to  distinguish  between  a  combining  and  an  active 
group,  and  the  same  terminology  will  be  adopted  throughout 
(agglutinoids,  complementoids,  etc.). 

The  change  of  toxin  into  toxoid  takes  place  in  all  exotoxins, 
but  at  very  different  rapidities.  Tetanolysin  is  transformed  com- 
pletely into  haemolysoid  in  a  day  or  so,  whilst  tetanospasmin,  the 
true  toxin  of  the  disease,  is  much  more  stable.  The  process  is 
accelerated  by  heat,  light,  and  the  access  of  oxygen,  and  by 
certain  chemical  substances  which  are  not  sufficiently  powerful 
to  destroy  the  toxin  outright.  Of  these  the  most  important  are 
a  solution  of  iodine  in  iodide  of  potassium,  and  bisulphide  of 
carbon. 

The  exotoxins  are  destroyed  outright  by  heating  to  the  boiling- 
point  (to  this  rule  there  are  a  few  exceptions,  none  of  which  has 
been  fully  examined),  by  strong  acids  and  alkalis,  and  by  the 
action  of  the  digestive  enzymes.  They  are,  as  a  rule,  precipitated 
by  the  substances  which  precipitate  proteids,  and  destroyed  by 
the  substances  that  destroy  those  bodies.  They  have,  further, 
the  power  of  attaching  themselves  to  precipitates,  of  whatever 
nature,  which  are  thrown  down  in  fluids  containing  them ;  so  that 
formerly  they  were  thought  to  be  albumins,  albumoses,  nucleo- 
albumins,  etc.,  since  they  were  carried  down  mechanically  when 
these  substances  were  precipitated  from  a  bacterial  culture  in 
which  they  were  present  along  with  the  exotoxin.  In  these  points 
again  they  closely  resemble  the  enzymes. 

They  are  substances  the  molecules  of  which  must  be  small  in 
comparison  with  those  of  the  coagulable  proteids,  since  they 
readily  pass  through  filters  (of  unglazed  porcelain  permeated  with 
gelatin)  which  retain  the  latter.  This  fact  was  put  to  an  ingenious 


48         TOXINS — ANALOGIES  WITH  ENZYMES 

use  by  Martin  and  Cherry  in  their  demonstration  that  diphtheria 
toxin  and  antitoxin  combine  chemically. 

Enzymes  are  also  substances  of  small  molecule,  and  pass 
through  similar  niters.  When  injected  into  suitable  animals 
enzymes  give  rise  to  the  production  of  anti-enzymes,  which  are 
exactly  equivalent  to  antitoxins.  Thus  we  see  that  in  many 
points  the  process  of  intoxication  with  the  bacterial  exotoxins 
presents  close  analogies  with  the  destruction  of  proteids,  etc.,  by 
enzymes;  and  to  these  we  might  add  the  suggestion  that  it  is 
very  probable  that  these  exotoxins  act,  partly  at  least,  by  a 
process  of  hydrolysis.  This  suggestion  is  based  partially  on  the 
fact  that  the  process  of  haemolysis  is  almost  certainly  one  of 
hydrolysis,  and  partially  on  the  appearance  of  poisoned  cells, 
which  look  as  if  they  had  absorbed  water  and  became  partly 
dissolved. 

There  is,  however,  one  feature  in  which  the  exotoxins  and 
their  allies,  the  bacterial  haemolysins,  are  absolutely  different  from 
the  enzymes.  In  the  case  of  the  enzymes  a  molecule  attaches 
itself  to  the  substance  to  be  attacked,  water  is  absorbed,  and  the 
whole  complex  molecule  breaks  down ;  and  in  this  process  the 
molecule  of  enzyme  is  set  free,  and  is  again  ready  to  attack 
another  molecule.  Thus  a  very  small  amount  of  the  active 
substance  can  decompose  a  large  amount  of  fermentable  substance. 
The  toxins  do  not  behave  in  this  way,  and,  as  far  as  we  know, 
a  molecule  of  toxin  which  has  united  with  one  molecule  of  proto- 
plasm is  never  set  free  to  attack  another.1  The  proof  of  this  is 
not  very  direct,  and  rests  mainly  on  the  fact  that  the  amount  of 
toxin  necessary  to  kill  two  animals  of  the  same  species  varies  roughly 
with  their  weight.  Thus  the  minimal  lethal  dose  of  diphtheria 
toxin  for  a  guinea-pig  of  250  grammes  will  not  kill  one  of  400. 
If  the  molecule  of  toxin  could  attack  one  molecule  of  cell 
substance  after  another  in  the  same  way  as  an  enzyme,  we  should 
expect  it  to  do  so,  though  after  a  longer  interval.  It  must  be 
confessed,  however,  that  this  proof  is  not  very  striking ;  excep- 
tions frequently  occur,  since,  as  a  rule,  older  animals  are  less 
susceptible  than  younger  ones  in  proportion  to  the  body-weight. 
But  it  is  certainly  true  with  regard  to  the  bacterial  haemolysins, 
since  we  can  test  them  on  the  same  sample  of  blood,  and  when 

1  It  may  possibly  undergo  dissociation,  and  be  set  free  to  attack  another 
molecule,  but  this  is  a  different  process :  the  molecule  first  attacked  is  not 
injured. 


ON    THE    NATURE    OF   TOXINS  4Q 

this  is  done  we  find  they  obey  the  law  of  multiple  proportions 
with  great  accuracy.  Thus  the  exotoxins  differ  from  the  enzymes 
mainly  in  the  fact  that  each  molecule  of  the  former  acts  once,  and 
once  only.  We  shall  subsequently  meet  another  group  of  sub- 
stances, of  very  similar  nature  but  of  animal  origin,  which  have 
an  enzyme-like  action,  but  are  destroyed  in  the  process.  They 
are  the  complements  (alexins,  etc.),  which  resemble  the  exotoxins 
in  many  respects,  and  might  well  be  called  the  animal  toxins. 

On  investigating  more  closely  the  action  of  the  exotoxins,  we 
find  that  certain  of  them  exert  their  pathogenic  action  mainly  on 
certain  cells  of  the  body.  The  most  marked  example  of  this  is 
in  tetanus,  which  practically  only  affects  the  cells  of  the  central 
nervous  system,  causing  in  them  definite  histological  changes, 
and  having  a  pharmacological  action  almost  exactly  like  that  of 
strychnine.  In  the  case  of  diphtheria  also  the  action  is  most 
marked  on  these  cells ;  this  is  best  shown  by  the  occurrence  of 
diphtheritic  paralysis  (associated  with  histological  changes  in  the 
ganglion  cells  similar  to  those  of  tetanus,  and  subsequent  degene- 
ration of  the  nerves),  which  occurs  after  the  action  of  minute 
doses  of  the  toxin.1  We  may  fairly  assume  that  when  but  an 
excessively  small  amount  of  toxin  is  present,  it  will  unite  with 
the  cells  with  which  it  has  most  affinity — in  this  case  with  those 
of  the  central  nervous  ganglia.  But  diphtheria  toxin  is  not 
limited  in  its  action,  as  tetanus  toxin  is,  and  can  act  upon  the 
tissue  cells  almost  without  exception.  Thus  we  find  that  the 
injection  of  a  large  dose  of  toxin  subcutaneously  is  followed  by 
the  production  of  an  acute  inflammatory  swelling,  showing  that 
it  can  poison  the  connective  tissues,  and  after  death  there  may  be 
focal  necrosis  of  the  liver,  degenerative  changes  in  the  renal 
epithelium,  fatty  degeneration  of  the  heart,  etc.,  showing  that 
the  toxin  may  act  on  all  these  organs  and  tissues.  We  may 
regard  it  as  a  good  example  of  a  general  protoplasmic  toxin 
having,  as  is  so  frequently  the  case,  a  special  action  on  certain 
cells.  The  toxins  of  most  diseases  come  under  this  heading,  the 
specialized  action  of  the  tetanus  toxin  being  unique. 

In  some  cases  we  can  study  the  action  of  the  exotoxins  and 
allied  substances  on  isolated  cells  in  vitro,  and  these  are  of  especial 
interest  from  the  ease  with  which  they  can  be  investigated,  and 
are  of  some  importance  in  disease.  They  are  the  leucolysins,  or 
leucotoxins,  and  the  haemolysins. 

1  If  we  accept  Arrhenius's  view  of  the  interaction  of  toxin  and  antitoxin. 

4 


50  THE    BACTERIAL    LEUCOLYSINS 

The  leucolysins  are  substances  which  are  formed  by  bacteria, 
and  which  have  the  power  of  killing  and  dissolving,  or  partially 
dissolving,  the  leucocytes  of  susceptible  animals.  Owing  to  the 
comparative  difficulty  of  obtaining  emulsions  of  living  leucocytes, 
they  have  not  been  submitted  to  the  same  thorough  examination 
as  have  been  the  bacterial  haemolysins ;  but  the  important  rela- 
tions between  the  leucocytes  and  immunity  lead  us  to*believe  that 
they  are  of  very  considerable  pathological  interest.  The  first 
to  be  described  was  that  formed  by  the  Streptococcus  pyogenes,  the 
action  of  which  on  the  living  leucocytes  was  shown  by  an  ingenious 
experiment  to  occur  in  vitro,  and  to  be  neutralized  by  means  of 
antileucolysin,  this  being  one  of  the  earliest  proofs  that  toxin  and 
antitoxin  form  a  chemical  combination,  and  that  the  preventive 
and  curative  effects  of  the  latter  are  not  due  to  some  profound 
influence  on  the  tissues  of  the  living  body,  by  which  they  are 
rendered  immune  before  the  toxin  can  attack  them.  The  method, 
invented  by  Neisser  and  Wechsberg,  is  based  on  the  fact  that 
living  leucocytes  have  the  power  of  deoxidizing  and  bleaching  a 
solution  of  methylene  blue.  When  a  solution  of  the  products  of 
growth  of  streptococci  is  added  to  an  emulsion  of  living  leuco- 
cytes, together  with  a  little  of  the  dye,  and  a  layer  of  liquid 
paraffin  added  to  prevent  the  further  access  of  air  and  subsequent 
oxidation  of  the  methylene  blue,  the  colour  no  longer  disappears, 
showing  that  the  leucocytes  have  been  killed.  If,  however,  a 
suitable  amount  of  antileucolysin  (obtained  by  injecting  the 
filtered  products  of  the  streptococci  into  an  animal)  be  added  to 
the  mixture  the  colour  disappears,  showing  that  the  leucocytes 
have  been  protected  from  the  action  of  the  leucolysin,  which  has 
now  been  neutralized  by  the  serum. 

The  action  of  the  leucolysins  can  also  be  studied  microscopi- 
cally in  vitro,  when  the  cells  are  seen  to  become  more  transparent, 
and  their  nuclei  to  become  more  indistinct,  and  ultimately  to 
disappear.  The  dissolving  leucocytes  look  very  much  like  those 
found  in  pus. 

Leucolysins  are  formed  by  the  Streptococcus  pyogenes,  the  staphy- 
lococcus,  and  B.  pyocyaneus,  and  probably  by  other  organisms. 

The  bacterial  hsemolysins  are  an  interesting  group  of  substances 
which  are  closely  allied  to  the  exotoxins  in  their  reactions,  but 
are  little  toxic,  if  at  all.  The  most  toxic  appears  to  be  that  of 
Streptococcus  pyogenes,  to  which  some  observers,  though  not  all, 
attribute  feeble  poisonous  powers  when  injected  into  animals. 


ON    THE    NATURE   OF   TOXINS  51 

At  the  same  time,  it  is  quite  certain  that  these  substances  play 
some  part  in  the  production  of  the  symptoms  of  various  diseases. 
The  anaemia  which  develops  so  rapidly  in  acute  sepsis  is  well 
known,  and  is  one  of  the  most  constant  symptoms  of  that  affec- 
tion ;  it  is  to  be  ascribed,  in  part  at  least,  to  the  destruction  of 
the  red  corpuscles  by  the  haemolysins  elaborated  by  the  strepto- 
cocci, staphylococci,  or  colon  bacillus,  if  these  happen  to  be  the 
infective  organisms.  The  blood  of  an  animal  which  has  been 
injected  with  virulent  streptococci  is  found  to  contain  haemolysin, 
and  that  this  is  actually  the  haemolysin  produced  by  the  strepto- 
coccus is  shown  by  the  fact  that  the  action  of  this  serum  is 
restrained  by  the  addition  of  serum  from  an  animal  treated  by 
injections  of  streptococcic  haemolysins.  Thus  it  is  proved  that  this 
organism  elaborates  its  haemolysin  in  vivo  as  well  as  in  vitro ;  and 
several  observers  have  found  that  it  is  those  species  of  strepto- 
coccus which  are  specially  virulent  to  animals  and  man  that 
form  haemolysins,  the  harmless  ones  doing  so  to  a  small  extent, 
if  at  all.  The  same  is  true  for  staphylolysins.  Further,  when 
a  culture  of  streptococci  which  is  but  slightly  virulent  and  forms 
but  little  haemolysin  is  rendered  more  virulent  by  "  passage  " 
through  rabbits,  its  power  of  forming  streptocolysin  is  increased. 

These  facts  render  it  certain  that  some  at  least  of  the  bac- 
terial haemolysins  act,  to  some  extent,  as  exotoxins,  though  the 
organisms  producing  them  certainly  form  other  and  more  im- 
portant specific  poisons.  We  may  consider  them  as  accessory 
toxins  of  comparatively  little  pathological  importance. 

The  similarity  in  nature  of  the  bacterial  haemolysins  and  the 
specific  exotoxins  is  shown  by  the  fact  that  (in  the  case  of  strep- 
tocolysin, and  probably  in  others)  they  can  become  converted  into 
htzmolysoids,  analogous  to  toxoids.  This  is  shown  as  follows : 
Streptocolysin  becomes  inert  in  a  week.  If  a  small  quantity  of 
blood-corpuscles  be  added  to  an  excess  of  this  inert  solution,  and 
then  thoroughly  washed  and  added  to  a  fresh  and  active  solution 
of  streptocolysins,  they  will  not  be  dissolved ;  the  corpuscles  had 
evidently  become  saturated  with  inert  haemolysoid,  and  are  now 
unable  to  take  up  any  haemolysin,  their  combining  powers  being 
satisfied  (see  Fig.  6). 

The  chief  bacterial  haemolysins  are  those  formed  by  the  tetanus 
bacillus,  the  staphylococcus,  the  Streptococcus  pyogenes,  the  B.  pyo- 
cyaneus,  B.  coli,  and  the  typhoid  bacillus.  Their  more  important 
features  will  be  recapitulated  briefly. 

4—2 


TETANOLYSIN   AND   STAPHYLOLYSIN 


Tetanolysin  is  formed  along  with  the  specific  toxin,  tetano- 
spasmin, when  the  B.  tetani  is  grown  in  broth,  the  two  substances 
being  formed  in  variable  amounts  under  different  circumstances. 
It  is  very  unstable,  disappearing  entirely  in  a  day  or  two  at  the 
room  temperature,  and  being  destroyed  by  heating  to  50°  C.  for 
twenty  minutes.  It  cannot  be  obtained  free  from  tetanospasmin, 
but  a  solution  of  tetanus  toxin  can  be  deprived  of  its  lysin,  and 
only  the  specific  toxin  left,  by  adding  some  red  corpuscles  to  the 
solution,  kept  at  a  low  temperature,  and  centrifugalizing  them 


FIG.  6. — A  "SATURATION  EXPERIMENT"  SHOWING  THAT  H^MOLYSOID  HAS 
THE  POWER  OF  COMBINING  WITH  RED  BLOOD-CORPUSCLES,  AND  SHIELD- 
ING THEM  FROM  THE  ACTION  OF  H^MOLYSIN.  (SCHEMATIC.) 

In  the  first  tube  the  corpuscles  are  shown  in  presence  of  an  excess  of  old 
or  heated  haemolysin ;  in  the  second  they  are  washed  clear  from  this 
excess,  and  are  apparently  unaltered  ;  in  the  third  active  haemolysin  is 
added,  but  the  corpuscles  are  not  dissolved,  as  they  would  be  in  a  control- 
tube  with  normal  corpuscles. 

off;  the  supernatural  fluid  will  contain  tetanospasmin,  whilst  the 
tetanolysin  will  have  combined  with  the  corpuscles. 

Staphylolysin  appears  in  alkaline  cultures  on  the  fourth  day, 
and  reaches  its  maximum  between  the  tenth  and  twelfth.  It  is 
an  unstable  substance,  but  more  stable  than  tetanolysin,  persist- 
ing for  a  fortnight  at  the  room  temperature,  and  requiring  a 
temperature  of  56°  C.  for  twenty  minutes  for  its  complete  destruc- 
tion— and  in  this  case  the  destruction  appears  to  be  really  com- 
plete, for  the  injection  of  the  heated  solution  is  said  not  to  lead 
to  the  production  of  an  antistaphylolysin.  Many  normal  sera, 
especially  those  of  man  and  the  horse,  contain  antistaphylolysin ; 


ON   THE   NATURE   OF   TOXINS  53 

perhaps  this  is  the  reason  why  slight  staphylococcic  infections  in 
man  are  not  associated  with  marked  haemolysis. 

Streptocolysin  is  formed  in  forty -eight  hours  when  a  virulent 
streptococcus  is  incubated  in  broth  containing  blood-serum  or 
ascitic  fluid,  and  it  is  a  remarkable  fact  that  the  nature  of  the 
serum  used  modifies  the  lysin  produced.  Thus  if  ox  serum  be 
employed  the  lysin  will  act  on  the  corpuscles  of  the  guinea-pig, 
rabbit,  and  man,  but  not  those  of  the  ox  or  sheep  ;  whilst  all 
these  will  be  dissolved  by  that  grown  in  broth  to  which  human 
serum  has  been  added. 

Streptocolysin  is  less  thermolabile  than  tetanolysin  and  staphy- 
lolysin,  requiring  an  exposure  of  ten  hours  to  55°  C.  or  of  two 
hours  to  70°  C.  for  complete  destruction. 

The  other  bacterial  haemolysins — i.e.,  those  produced  by  the 
B.  pyocyanens,  B.  typhosus,  and  B.  coli  —  are  quite  different  from 
the  foregoing  in  being  thermostable.  Thus  pyocyanolysin  re- 
sists a  temperature  of  120°  C.  for  thirty  minutes.  Typholysin 
appears  to  be  less  resistant,  but  is  definitely  thermostable. 
Colilysin  is  as  stable  as  pyocyanolysin  ;  it  is  not  destroyed  by 
a  temperature  of  120°  C.  for  half  an  hour,  and  does  not  undergo 
spontaneous  weakening  for  months.  It  is  obvious  that  these 
substances  are  different  in  character  from  the  other  haemolysins 
and  exotoxins,  and  the  fact  that  (in  the  case  of  pyocyanolysin  and 
colilysin,  the  most  heat-resistant  of  the  group)  the  haemolytic 
property  of  the  culture  only  appears  when  it  becomes  strongly 
alkaline  and  is  roughly  parallel  in  degree  to  the  amount  of  alka- 
linity, has  led  some  to  think  that  the  substances  are  not  the  true 
haemolysins  at  all,  but  merely  simple  alkaline  chemical  products 
of  growth  ;  and  this  is  corroborated  by  the  fact  that  much  of  the 
haemolytic  power  is  taken  away  on  neutralization  with  a  weak 
acid.  It  appears  that  this  is  not  the  case,  since  in  a  culture  of 
B.  coli  at  a  temperature  of  23°  C.,  the  alkalinity  reaches  its  maxi- 
mum on  the  fifth  day,  whilst  the  haemolytic  property  does  not 
appear  until  later.  The  subject  requires  further  investigation,  and 
at  present  it  is  advisable  to  disregard  these  substances  which  differ 
so  much  from  their  allies. 

The  chemical  nature  of  the  exotoxins  has  been  the  subject  of 
much  controversy,  and  is  still  very  imperfectly  understood.  It 
will  not  be  discussed  at  great  length,  since  from  the  point  of 
view  of  immunity  it  is  not  of  very  great  importance. 

The  close  analogy  between  the  bacterial  exotoxins  and  certain 


54  CHEMICAL   NATURE   OF   TOXINS 

vegetable  toxins,  such  as  ricin  and  abrin  (which  were  thought  to 
be  definitely  proteid  in  nature),  led,  very  early  in  the  history  of 
the  subject,  to  the  view  that  these  toxins  are  proteid  in  nature, 
and  this  view  was  strengthened  by  the  fact  that  when  the  diph- 
theria or  tetanus  bacillus  is  grown  in  an  albuminous  fluid,  proteid 
substances  which  are  toxic  and  give  the  specific  reactions  of  the 
toxins  in  question  can  be  precipitated  therefrom.  Thus  Hankin 
and  Sidney  Martin  found  toxic  albumoses  in  bacterial  cultures, 
and  apparently  succeeded  in  proving  that  abrin  is  an  albumose. 
Brieger  isolated  a  toxalbumin  from  diphtheria  cultures,  and 
Sidney  Martin  showed  that  from  cultures  of  the  same  organism 
in  alkali  albumin  it  is  possible  to  prepare  an  albumose  which 
he  thought  to  be  the  specific  toxin.  Many  similar  researches 
were  published,  and  the  exotoxins  were  regarded  as  being  albu- 
minoid in  nature,  and  the  term  toxoprotein  was  applied  to  them. 
Several  writers — Duclaux  in  particular — argued  that  this  was 
not  the  case,  and  thought  that  these  proteid  substances  merely 
carried  the  true  toxins  with  them  mechanically  on  precipitation, 
just  as  the  precipitates  of  inert  substances  such  as  cholesterin 
will  carry  enzymes  down  with  them.  This  theory  was  sup- 
ported by  Brieger  and  Cohn,  who  purified  tetanus  toxin  from  all 
ordinary  proteids,  and  especially  by  the  researches  of  Buchner 
and  Uschinsky,  who  cultivated  tetanus  and  diphtheria  bacilli  in 
solutions  devoid  of  all  albuminous  material,  the  necessary  nitro- 
genous nutriment  being  provided  by  asparagin.  Under  these 
circumstances  the  toxic  solution  contains  neither  albumoses, 
peptones,  nor  known  proteids  of  any  description.  The  toxins 
thus  formed  are  present  in  infinitesimally  small  amount,  and  have 
never  been  obtained  in  a  pure  form,  nor  submitted  to  ultimate 
analysis.  It  is  known,  however,  that  they  contain  nitrogen,  that 
they  are  readily  destroyed  by  heat,  and  that  they  are  dialysable. 
These  considerations  lead  us  to  the  supposition  that  they  are 
closely  allied  to  the  proteids,  and  especially  to  the  albumoses  or 
peptones,  but  form  a  group  differing  from  any  of  them,  and 
approximating  more  closely  to  the  enzymes.  That  this  is  the 
case  appears  certain  from  the  facts  brought  out  by  researches  on 
the  antibodies  ;  all  the  substances  of  known  chemical  composition 
which  lead  to  the  production  of  antibodies  on  injection  into  suit- 
able animals  are  either  proteids  or  else  substances  of  indefinite 
composition  similar  to  the  toxins,  and  apparently  all  proteids  will 
lead  to  the  production  of  antibodies  on  injection  into  suitable 


ON    THE    NATURE    OF   TOXINS  55 

animals.  These  facts  lead  us  to  the  belief  that  the  exotoxins  are, 
at  any  rate,  allied  to  the  proteids,  and  form  with  the  enzymes  a 
group  of  the  substances  of  peculiar  composition. 

We  have  referred  above  to  ricin  as  a  substance  once  thought 
to  be  of  definite  proteid  nature,  and  a  few  facts  may  be  given 
concerning  this  substance,  which  is  closely  allied  in  every  way 
to  the  bacterial  toxins,  and  which  may  be  taken  as  a  type  of  the 
vegetable  toxins  or  phytotoxins.  It  occurs  in  the  seeds  of  various 
species  of  Ricinus,  and  was  formerly  regarded  as  being  a  proteid, 
since,  like  the  bacterial  toxins,  it  is  carried  down  mechanically 
with  proteid  precipitates.  Thus  Stillmarck  regarded  it  as  a  globulin, 
since  he  prepared  it  from  the  seeds  by  a  process  which  was  adapted 
to  the  separation  of  those  substances  (solution  in  10  per  cent.  NaCl, 
precipitation  with  sodium  or  magnesium  sulphate,  and  dialysis). 
But  Jacoby  thought  he  had  succeeded  in  separating  it  entirely 
from  its  proteid  accompaniment,  making  use  of  the  fact  that  when 
a  mixture  of  ricin  and  the  other  substances  present  in  the  seeds 
are  acted  on  by  trypsin,  the  active  principle  is  acted  on  but  slightly, 
if  at  all ;  the  ricin  itself,  in  a  pure  state,  is  readily  digested  by 
trypsin,  like  the  other  toxins.  Jacoby  digested  an  extract  of 
castor-oil  seeds  for  five  weeks,  and  then  added  enough  ammonium 
sulphate  to  render  the  fluid  60  per  cent,  saturated,  and  ricin  was 
thrown  down  in  an  almost  pure  state  ;  it  was  purified  by  repreci- 
pitation,  and  then  found  not  to  give  any  of  the  proteid  reactions, 
though  it  retained  the  characteristic  toxic  properties  of  the  sub- 
stance. Quite  recently,  however,  Osborne,  Mendel,  and  Harris 
obtained  ricin  in  a  very  pure  form,  and  found  it  to  be  either 
proteid  in  nature  or  at  least  inseparably  associated  with  coagulable 
albumin ;  its  toxicity  was  removed  by  tryptic  digestion  or  heat 
coagulation.  Its  great  potency  (TTrV^  mgr-  being  a  lethal  dose 
per  i  kilo  of  rabbit)  suggests  that  the  substance  which  they 
prepared  was  really  pure. 

Ricin  resembles  the  bacterial  toxins  in  the  following  points : 
It  has  a  period  of  incubation  ;  it  gives  rise  to  an  antitoxin  when 
suitably  administered ;  it  is  extraordinarily  potent,  the  lethal  dose 
per  kilo  of  weight  (in  rabbits)  being  a  minute  fraction  of  a 
gramme  ;  it  is  destroyed  by  boiling  ;  and  it  is  much  less  potent  on 
ingestion  than  on  injection.  Its  main  toxic  properties  are  fever, 
loss  of  weight,  albuminuria,  haematuria,  and  haemorrhage  from 
the  intestine  ;  death  occurs  in  about  twenty-four  hours  with  acute 
nervous  symptoms.  It  has  a  most  interesting  and  characteristic 


56  THE    ENDOTOXINS 

action  on  the  blood,  clumping  the  corpuscles  in  a  peculiar  way, 
even  at  a  dilution  of  i  :  600,000,  and  also  haemolyzing  them. 

Further  research  leads  us  to  believe  that  the  toxin  molecule 
may  be,  and  under  ordinary  circumstances  is,  actually  of  more 
complex  constitution,  being  combined  with  a  molecule  of  true 
proteid.  We  have  already  pointed  out  the  fact  that  streptocolysin 
differs  in  its  reactions  according  to  the  origin  of  the  serum  on 
which  it  is  grown.  The  best  example,  however,  is  derived  from 
diphtheria  toxin  when  grown  in  broth  containing  blood-serum  or 
plasma,  and  subsequently  heated.  This  solution  is  but  feebly 
toxic,  probably  from  the  toxins  having  undergone  a  change  into 
toxoids,  yet  it  possesses  the  power  of  immunizing  an  animal 
against  diphtheria,  and  of  stimulating  the  production  of  anti- 
toxin to  an  unusual  degree,  but  only  on  condition  that  it  is 
injected  into  an  animal  of  the  same  species  as  that  from  which 
the  serum  in  which  the  bacillus  grew  was  obtained.  Thus  horse- 
serum  toxin  will  stimulate  the  production  of  antitoxin  in  horses, 
but  not  in  goats  or  rabbits,  and  so  forth.  We  are  justified  in  sup- 
posing that  the  essential  toxin  molecule  formed  by  the  diphtheria 
bacillus  exists  in  this  fluid  in  a  state  of  combination  with  a  specific 
proteid  of  horse  serum,  and  that  the  resulting  compound  molecule 
differs  from  that  form  when  the  bacillus  is  grown  in  goat  serum, 
in  which  the  essential  toxic  molecule  is  united  with  a  different 
proteid.  We  may  suppose  that  this  essential  toxin  molecule  is 
produced  in  Buchner  and  Uschinsky's  asparagin  solution,  but  that 
it  is  not  produced  under  ordinary  conditions,  being  in  a  state  of 
combination  with  proteid  materials  of  more  complex  structure. 
These  facts  render  further  research  into  the  chemical  nature  of 
the  exotoxins  of  comparatively  little  importance. 

THE  ENDOTOXINS. 

In  the  case  of  diphtheria  and  tetanus  and  a  few  other  organisms 
the  mode  of  formation  of  toxins  is  a  perfectly  simple  one,  and 
one  exactly  analogous  to  the  formation  of  soluble  enzymes.  In 
most  other  cases,  however,  the  facts  are  less  easy  to  understand, 
and  seem  to  point  to  the  formation  of  a  toxin  which  remains 
under  normal  circumstances  locked  up  in  the  substance  of  the 
bacteria,  just  as  invertase  and  diastase  are  contained  within  the 
yeast  cell,  and  not  excreted  by  it  into  the  surrounding  fluid.  A 
satisfactory  theory  as  to  the  nature  of  these  toxins  is  not  forth- 
coming, and  the  experimental  results  obtained  by  various 


ON   THE    NATURE    OF  TOXINS  57 

observers  is  very  contradictory  and  difficult  to  understand,  the 
difficulty  being  increased  by  the  fact  that  in  the  earlier  researches 
the  distinction  between  antitoxic  and  bacterial  immunity  was  not 
understood.  As  a  result  of  this  we  have  to  be  careful  in  in- 
terpreting these  early  results,  so  as  to  make  sure  that  when  the 
author  speaks  of  a  serum  as  containing  antitoxin  he  does  not 
really  mean  that  it  contains  a  protective  substance  which  may 
not  be  an  antitoxin  at  all.  In  many  cases  the  data  are  not 
sufficient  for  us  to  discover  its  actual  nature. 

The  organisms  on  which  the  chief  amount  of  experimental 
work  has  been  done  are  those  of  cholera,  typhoid,  tubercle, 
anthrax,  and  the  pneumococcus,  and  it  is  these  which  we  shall 
discuss  in  chief,  excluding,  however,  the  consideration  of  the 
toxins  of  the  tubercle  bacillus  for  separate  consideration  in  a 
subsequent  chapter. 

The  general  facts  brought  out  by  experiments  with  organisms 
such  as  those  of  typhoid  and  cholera  are  these :  The  germ-free 
filtrate  of  a  young  and  actively  growing  culture  is  very  slightly 
toxic,  if  at  all.  The  nitrate  of  an  older  culture  is  usually  feebly 
toxic,  but  to  a  degree  which  can  hardly  be  compared  with  that  of 
diphtheria  or  tetanus  ;  it  may  take  several  cubic  centimetres  to 
kill  a  rabbit  or  guinea-pig.  And  even  this  feeble  toxicity  is 
largely  discounted  by  the  fact  that  the  nitrate  may  contain  acids, 
nitrites,  etc.,  which  are  poisonous,  but  in  no  way  related  to  true 
toxins.  Yet  in  some  cases  exotoxins  do  exist  in  the  filtrate, 
since  it  is  possible  to  obtain  an  antitoxin  for  them.  The  re- 
actions of  these  antitoxins,  however,  are  peculiar,  in  that  the  law 
of  multiples  does  not  seem  to  apply  beyond  a  certain  figure.  This 
is  well  seen  in  the  case  of  B.  pyocyaneus,  which  forms  a  sort  of 
connecting-link  between  cholera  and  diphtheria,  in  that  it  forms 
a  definite  though  feeble  exotoxin,  whilst  the  immunity  to  it  is 
bactericidal.  Wassermann  showed  that  it  is  possible  to  produce 
a  true  antitoxin  against  this  toxin,  and  to  determine  the  amount 
which  will  just  neutralize  one  lethal  dose.  He  found,  however, 
that  a  multiple  of  this  amount  of  antitoxin  beyond  ten  would  not 
protect  an  animal  against  a  corresponding  dose  of  toxin,  With 
larger  doses  of  toxin  even  a  great  excess  of  antitoxin  was  power- 
less to  prevent  a  lethal  issue.  Similar  results  have  been  obtained 
in  the  case  of  cholera.  These  and  other  results  have  led  some 
authorities  to  consider  that  these  exotoxins  are  not  the  specific 
toxins  which  the  pathogenic  action  of  the  bacillus  defends,  but 


58          MACFADYEN'S  RESEARCHES 

secondary  products  of  but  little  importance.  Thus,  in  the  case  of 
B.  pyocyaneus  it  is  possible  to  immunize  an  animal  by  cautious 
injections  of  living  organisms,  yet  its  serum  has  no  antitoxic 
powers  against  the  so-called  toxin. 

These  facts  have  turned  attention  to  the  bodies  of  the  bacteria 
themselves,  with  the  result  that  they  have  been  found  to  be 
definitely  toxic,  although  in  many  cases  the  toxicity  is  not  great. 
The  theory  has  therefore  been  put  forward — by  Pfeiffer  espe- 
cially— that  under  normal  circumstances  these  organisms  do  not 
secrete  a  soluble  toxin,  but  that  their  protoplasm  itself  is  toxic, 
and  that  it  is  only  set  free  on  the  death  and  solution  of  the  cell, 
thus  accounting  for  the  slight  toxicity  of  old  cultures,  in  which 
such  a  solution  of  the  cells  must  take  place.  The  symptom  of 
the  disease  caused  by  these  organisms  is  attributed  to  the  solu- 
tion of  the  bacteria  by  the  fluids  of  the  body. 

The  study  of  these  endotoxins  has  not  left  the  matter  clear. 
They  are  present  in  the  bodies  of  the  bacteria,  whether  the  latter 
have  been  killed  by  heat,  by  antiseptics,  or  by  drying.  They  are 
apparently  but  slightly  soluble  in  water,  but  can  be  obtained  in 
solution  by  autolysis  of  the  bacteria  in  normal  saline  solution  in 
the  incubator,  by  grinding  the  dead  bacilli,  or  by  the  use  of  very 
high  pressure  (the  method  introduced  by  Buchner  for  the  extrac- 
tion of  endo-enzymes  from  yeast).  But  it  did  not  appear  possible 
to  produce  an  antitoxin  against  this  poisonous  material ;  in 
addition,  animals  which  have  been  immunized  against  the  living 
organism  might  be  as  susceptible  as  before  to  the  dead  bacteria, 
or  to  extracts  of  them. 

The  researches  of  Macfadyen  and  Rowland  have  apparently 
disproved  this,  and  tend  to  support  the  opinion  that  the  endo- 
toxin  is  a  true  toxin,  for  which  an  antitoxin  can  be  obtained. 
They  obtained  young  cultures  of  various  organisms,  froze  them 
at  the  temperature  of  liquid  air,  and  then  ground  them  (whilst 
solid)  into  an  impalpable  powder.  This  was  made  into  a  paste 
with  normal  saline  solution  and  centrifugalized,  to  remove  any 
solid  particles.  The  juice  thus  obtained  was  sterile.  It  was 
more  powerful  than  endotoxins  prepared  in  other  ways,  and  it 
acted  very  quickly,  having  a  very  short  period  of  incubation,  if 
any.  Thus,  in  the  case  of  the  typhoid  toxin  i  c.c.  killed  in  three 
hours  and  J^  c.c.  in  less  than  two  days,  on  intraperitoneal  in- 
jection. It  was  less  active  on  subcutaneous  injection — not  more 
so,  in  fact,  than  other  toxins  of  the  typhoid  bacillus — requiring 


ON   THE    NATURE   OF   TOXINS  59 

£  to  y1^  c.c.  to  kill  in  seven  days.  Macfadyen  and  Rowland 
found  that  they  could  immunize  animals  against  their  toxin,  and 
that  its  serum  was  antitoxic.  These  researches  are  difficult  to 
harmonize  with  those  of  other  observers.  We  must  admit,  how- 
ever, that  it  is  possible  to  prepare  an  antitoxin  to  the  endotoxins. 
The  failure  of  other  observers  to  do  so  may  be  owing  to  the  fact 
that  their  toxins  were  not  prepared  in  so  suitable  a  manner  for 
this  purpose,  and  may  have  undergone  some  unknown  secondary 
alterations. 

But  these  researches  do  not  clear  up  the  whole  of  the  mystery, 
for  some  observations  of  Metchnikoff  and  others  show  that  the 
V.  cholera:  can  produce  a  soluble  toxin  whilst  in  the  animal,  and 
apparently  without  being  killed  in  the  process.  These  observers 
prepared  collodion  bags,  which  they  filled  with  cultures  of  this 
organism,  hermetically  sealed,  and  inserted  into  the  peritoneal 
cavities  of  guinea-pigs.  The  animals  died  in  a  few  days  with 
the  symptoms  of  cholera  intoxication,  although  no  bacteria  had 
escaped  from  the  sacs ;  the  organisms  in  that  situation  were  still 
alive.  Control  experiments  with  dead  organisms  showed  that 
little  toxin  was  present ;  the  animals  remained  alive,  though  they 
might  show  some  symptoms  of  toxic  action.  It  appears,  there- 
fore, that  the  living  bacteria  do  elaborate  an  exotoxin  whilst 
within  the  animal  body,  and  that  this  exotoxin  has  the  power  of 
diffusing  through  a  collodion  membrane.  Welch  has  suggested 
an  explanation  which  cannot  be  discussed  fully  here,  but  which 
may  be  mentioned  briefly.  He  points  out  that  when  bacteria 
are  injected  in  living  animals  the  tissues  of  the  latter  react  and 
produce  substances — bacteriolysins,  etc. — which  are  injurious  to 
the  bacteria,  and  which  determine  in  part  the  resistance  of  the 
host,  and  suggests  that  the  bacteria  may  also  react  in  a  similar 
way  to  the  cells  with  which  they  are  brought  in  contact.  Just  as 
the  animal  host  only  produces  its  toxins — the  bactericidal  sub- 
stances— when  the  bacteria  are  brought  into  contact  with  it,  so 
the  bacteria  may  only  produce  their  protective  substances — the 
unidentified  true  toxins — when  brought  into  contact  with  aggressive 
animal  cells.  If  this  is  the  case  it  is  obvious  that  we  cannot  expect 
to  produce  these  toxins  in  vitro,  except  perhaps  by  cultivation  of 
the  bacteria  in  question  in  fresh  serum  from  an  immunized  animal. 


CHAPTER  III 

THE  PHENOMENA  OF  ANTITOXIN 
FORMATION 

As  a  general  rule,  to  which  there  are  important  exceptions,  it  is 
necessary  to  make  use  of  susceptible  animals  for  the  production 
of  antitoxin.  When  toxin  is  injected  into  animals  in  which  it  pro- 
duces no  injurious  effects,  it  either  disappears  rapidly  from  the 
blood  or  remains  for  a  long  time  in  that  fluid  or  in  the  tissues 
without  leading  to  the  formation  of  antitoxin.  The  most  remark- 
able exception  to  this  rule  is  the  way  the  cayman  reacts  to  tetanus 
toxin.  The  animal  is  immune,  and  if  kept  in  the  cold  (20°  C.)  the 
toxin  soon  disappears  from  the  blood,  no  antitoxin  being  formed.  If, 
however,  it  is  kept  at  an  elevated  temperature  (32°  to  37°  C.),  the 
toxin  disappears  as  before,  but  now  antitoxin  makes  its  appearance 
(Metchnikoff).  Such  cases  are  exceptional,  and  when  we  wish 
to  procure  antitoxin,  we  make  use  of  an  animal  in  which  the 
toxin  in  question  produces  symptoms  of  intoxication.  The  pro- 
cess is  usually  much  easier  in  large  animals,  such  as  horses  or 
goats,  than  in  small  ones,  such  as  rabbits  or  guinea-pigs,  the 
immunization  of  which  presents  considerable  difficulties.  We 
shall  take  as  illustrations  of  the  general  phenomena  of  the  pro- 
cess the  methods  adopted  for  procuring  diphtheria  antitoxin  and 
tetanus  antitoxin  from  horses,  since  these  have  become  so  familiar 
from  their  extensive  application. 

On  injecting  a  small  dose  of  a  potent  diphtheria  toxin  sub- 
cutaneously  into  a  horse — say,  \  c.c. — under  the  skin  of  the 
neck  we  find  there  is  a  latent  interval  of  a  few  hours  or  a 
day  before  the  development  of  symptoms ;  then  there  is  a  l»cel 
reaction,  consisting  in  the  formation  of  a  hard  brawny  mass  ©f 
inflammatory  oedema  round  the  site  of  the  inoculation,  and  a 
general  reaction,  consisting  in  fever,  anorexia,  and  symptoms  of 
general  malaise.  These  symptoms  last  a  day  or  two,  according 
to  the  dose  of  toxin  injected,  its  potency,  and  the  degree  of  sus- 

60 


THE    PHENOMENA   OF   ANTITOXIN    FORMATION  6l 

ceptibility  of  the  animal ;  and  when  they  have  passed  off  a  small 
amount  of  antitoxin  will  be  found  in  the  blood,  and  the  animal 
will,  as  a  rule,  be  found  to  be  less  susceptible  to  the  action  of  the 
toxin  than  before,  so  that  the  injection  of  the  same  dose  will 
produce  less  reaction,  both  local  and  general. 

This,  however,  is  not  always  the  case,  and  careful  research 
leads  us  to  the  belief  that  the  appearance  of  immunity  is  preceded 
by  a  period  of  hypey  sensitiveness,  in  which  the  animal  betrays  a 
greatly  increased  susceptibility  to  the  action  of  the  toxin,  and  this 
in  spite  of  the  fact  that  it  may  contain  quite  large  quantities  of 
antitoxin  in  its  blood.  Thus  it  happens  not  infrequently  that 
after  a  horse  has  passed  successfully  through  the  early  stages 
of  immunization  to  diphtheria  toxin,  and  has  developed  far  more 
antitoxin  than  is  necessary  to  neutralize  the  doses  of  toxin  with 
which  it  is  being  treated,  it  yet  will  die  after  the  injection  of  an 
amount  which  it  would  appear  must  be  immediately  rendered  inert 
as  soon  as  it  came  into  contact  with  the  plasma.  Such  cases  have 
been  reported  from  the  Pasteur  Institute,  Behring  and  Kitashima, 
and  others,  and  by  Brieger  for  tetanus.  In  the  latter  an  immunized 
horse  died  after  an  injection  of  tetanus  toxin  with  the  typical 
symptoms  of  tetanus  intoxication,  and  after  death  its  blood  con- 
tained much  free  antitoxin.  The  phenomenon  has  probably  been 
witnessed  by  most  observers  who  have  been  engaged  in  the  manu- 
facture of  antitoxin,  though  it  has  become  much  less  frequent  since 
the  introduction  of  modern  methods  for  the  early  treatment  of 
animals.  It  is  an  exceedingly  puzzling  one,  and  we  shall  leave 
its  further  interpretation  until  later ;  here  it  is  sufficient  to  say 
that  Behring's  theory  of  the  occurrence  of  a  stage  in  which  the 
tissues  are  hypersensitive  to  the  toxin  is  well  established. 

The  difficulty  of  immunizing  the  small  animals  of  the  laboratory 
to  these  toxins  appears  to  depend  in  large  measure  on  the  marked 
development  of  hypersensitiveness.  Thus  Behring  and  Kitashima 
found  that  they  could  kill  a  guinea-pig  with  ^J^  of  the  "  minimal 
lethal  dose"  of  tetanus  toxin,  if  this  amount  were  divided  into 
several  doses  and  given  at  suitable  intervals,  and  similar  facts 
have  been  recorded  by  others. 

The  most  striking  proof  of  the  occurrence  of  hypersensitiveness 
in  the  process  of  immunization  has  been  investigated  by  Behring, 
who  pointed  out  that  normal  horses  show  no  local  effects  from  the 
injection  of  small  quantities  of  tetanus  toxin ;  their  connective 
tissues  are  insusceptible  to  its  action.  As  the  animal  becomes 


62  CHOICE   OF   TOXINS 

immunized  to  the  action  of  the  toxin  this  is  not  the  case;  the 
tissues  at  the  site  of  inoculation  react  to  the  poison  with  the  pro- 
duction of  a  mass  of  inflammatory  redema  similar  to  that  seen  in 
a  horse  injected  with  diphtheria  toxin.  It  is  obvious  that  these 
connective  tissues  have  become  more  susceptible  to  the  action  of 
the  tetanus  toxin,  and  this  in  spite  of  the  antitoxin  with  which 
they  are  bathed. 

In  order  to  avoid  the  difficulties  arising  from  the  occurrence  of 
hypersensitiveness  in  the  early  stages  of  immunization,  the  use 
of  unaltered  toxin  has  now  been  practically  abandoned,  the  follow- 
ing methods,  either  alone  or  in  combination,  being  employed 
instead : 

1.  The  use  of  mixtures  of  toxin  and  antitoxin,  the  latter  being 
present  in  amount  sufficient  to  neutralize   all   the  toxin,  or  in 
excess.     This  is  repeated  several  times,  the  amount  of  antitoxin 
given  being  gradually  reduced,  until  at  last  a  small  amount  of 
unaltered  toxin  is  given. 

It  must  not  be  thought  that  the  immunity  which  is  acquired  in 
this  case  is  simply  passive,  and  due  to  the  free  antitoxin  which  is 
injected.  The  process  is  probably  fundamentally  different.  We 
shall  revert  to  it  subsequently. 

2.  The  injection  of  toxoids.     This  method  is  of  especial  advan- 
tage in  the  case  of  tetanus,  to  which  toxin  animals  are  extremely 
sensitive,  and  the  dangers  of  the  early  stages  of  the  process  of 
immunization  are  very  great.     The  toxin  formed  in  the  cultures 
may  be  transformed  into  toxoids  by  the  action  of  trichloride  of 
iodine,  a  solution  of  iodine  in  iodide  of  potassium,  or  by  heat, 
the  filtered  cultures  being  exposed  to  a  temperature  of  60°  C.  for 
a  time  sufficient  to  destroy  their  toxicity.     Toxin  that  has  been 
heated  to  a  temperature   much  higher  than   this  is  completely 
destroyed,  and  is  useless  for  the  process. 

3.  The  use  of  serum  toxin,  which  probably  contains  toxoids  in 
an  unusual  condition  of  activity.     This  method  was  introduced 
by  Cartwright  Wood,  and  is  now  in  general  use  in  this  country 
for  a  part  at  least  of  the  process  of  immunization,  since  it  leads  to 
a  more  rapid  production  of  antitoxin  of  high  potency  than  can  be 
obtained  by  other  methods  in  the  same  time.     Ordinary  alkaline 
broth  is  inoculated  with  diphtheria  bacilli,  and  incubated  for  a  week 
at  37°  C.     Then  15  to  30  per  cent,  of  its  volume  of  serum  from  an 
animal  of  the  same  species  as  is  to  be  used  in  the  process  of 
immunization  is  added,  and  the  incubation  continued  for  a  month 


THE    PHENOMENA   OF   ANTITOXIN    FORMATION  63 

or  six  weeks.  It  is  then  heated  to  65°  C.  for  half  an  hour  and 
filtered.  It  gives  rise  to  marked  febrile  reaction  and  but  little 
local  reaction.  The  initial  dose  is  200  to  300  c.c. 

In  giving  these  large  doses  the  most  convenient  method  is  to 
use  a  large  wash-bottle,  the  side  of  which  is  graduated  in 
cubic  centimetres.  To  the  outflow  arm  there  is  attached  2  or 
3  yards  of  pressure  tubing,  in  the  farther  end  of  which  a  strong 
exploring  needle  is  inserted,  and  firmly  wired  in  place.  The 
pressure  is  obtained  by  means  of  a  bicycle  pump  attached  to  the 
inflow  tube  of  the  wash-bottle  by  means  of  pressure  tubing. 
There  should  be  a  lateral  branch  communicating  with  a  mano- 
meter, by  which  the  pressure  can  be  regulated.  Very  high 
pressure  is  sometimes  necessary,  especially  in  the  later  stages  of 
the  process,  when  the  subcutaneous  tissues  of  the  horse's  neck 
become  sclerosed  and  dense  from  the  repeated  injections.  The 
apparatus  is  most  easily  sterilized  by  passing  strong  carbolic 
lotion  through  it. 

On  testing  the  blood-serum  from  time  to  time,  it  is  found  that 
the  amount  of  antitoxin  gradually  rises,  each  injection  being 
followed  by  an  increase  in  the  antitoxic  value  of  the  serum.  Thus 
the  process  is  a  cumulative  one,  the  antitoxic  level  being  raised 
step  by  step  until  a  certain  height  is  reached.  This  height  differs  in 
different  animals.  Thus  Atkinson,  in  summarizing  his  experience 
of  100  horses,  found  that  half  of  this  number  gave  less  than 
300  units  of  antitoxin  per  cubic  centimetre,  a  quarter  between 
300  and  500,  whilst  three  gave  more  than  800.  There  appears 
to  be  no  method  of  investigation  by  which  the  value  of  a  horse 
as  a  source  of  antitoxin  can  be  predicted  early  in  the  course 
of  treatment,  and  the  great  variability  amongst  different  animals 
is  probably  the  reason  that  different  observers  have  come  to 
such  divergent  opinions  as  to  the  best  doses  to  give  and  the 
most  suitable  intervals  between  each.  Here  are  three  chief 
methods : 

(a)  By  the  use  of  large  doses  of  toxin,  250  to  500  c.c.  every  day, 
or  almost  every  day,  leaving  an  interval  of  a  week  or  ten  days 
before  the  bleeding,  so  as  to  allow  the  last  injection  to  produce  its 
maximum  effect. 

(b)  The  use  of  large  injections  (similar  to  the  former)  at  longer 
intervals — five  to  ten  days. 

(c)  The  use  of  relatively  small  doses  of  weak  toxins  repeated 
every  day. 


64  THE    NEGATIVE    PHASE 

All  these  methods  have  their  advocates,  and  good  results  can 
apparently  be  obtained  by  all. 

On  ceasing  to  inject  toxin,  it  usually  happens  that  the  antitoxic 
value  of  the  serum  commences  to  decline,  and,  in  the  absence  of 
further  injections,  would  probably  continue  to  do  so  until  it  had 
entirely  disappeared  from  the  blood.  In  a  few  cases,  however, 
a  period  of  antitoxic  equilibrium  is  maintained  for  some  time,  the 
amount  of  antitoxin  lost  by  the  excretions  or  destroyed  in  the 
system  being  compensated  for  by  a  fresh  production  of  the  same 
amount.  When  this  is  the  case  the  phenomena  resulting  from 
the  injection  of  a  single  dose  of  toxin  can  be  traced  with  ease,  and 
is  of  great  importance,  as  will  appear  subsequently.  The  first 
effect  of  the  injection  is  the  production  of  a  negative  phase,  in  which 
the  amount  of  antitoxin  in  the  blood  is  suddenly  and  greatly 
diminished.  This  production  of  a  negative  phase  is  apparently 
a  general  phenomenon,  and  is  found  to  occur  in  the  development 
of  nearly  all  antibodies  in  which  it  has  been  investigated.  If  the 
dose  of  the  primary  substance  (toxin,  etc.)  is  very  small,  the 
negative  phase  may  be  short  in  duration  and  very  slight  in  extent, 
and  may  be  overlooked,  or  may  possibly  be  omitted  altogether. 
Its  explanation  is  very  uncertain  and  cannot  be  discussed  here, 
but  it  must  be  pointed  out  that  it  is  not  due  to  the  neutralization 
of  the  antitoxin  in  the  blood  by  the  toxin  injected ;  the  proof  of 
this  is  that  it  is  large  out  of  all  proportion  to  the  latter.  Thus  in 
one  reported  case  the  fall  in  antitoxic  value  of  the  serum  which 
occurred  in  the  negative  phase  would  have  required  an  injection 
of  toxin  12,000  times  as  large  as  was  actually  given  if  it  were  due 
to  simple  neutralization.  The  length  of  the  negative  phase  varies 
in  different  animals,  and  can  only  be  learnt  by  experiment.  It 
appears  to  be  roughly  proportional  (in  the  same  animal)  to  the 
amount  of  primary  substance  injected  :  the  larger  the  doses  of 
toxin,  the  greater  the  fall  and  the  longer  its  duration.  It  is, 
of  course,  synchronous  with  the  toxic  symptoms,  if  any,  of  the 
substance  injected,  since  both  are  due  to  the  action  of  this  sub- 
stance on  the  blood  and  tissues  ;  but  the  two  do  not  appear  to  be 
mutually  dependent :  a  well-marked  negative  phase  may  appear 
without  any  other  symptoms  of  disease. 

The  negative  phase  is  succeeded  by  a  rise,  the  positive  phase, 
in  which  the  antitoxic  value  of  the  blood  reaches  and  usually 
surpasses  its  previous  level.  It  commonly  reaches  its  maximum 
in  about  a  week,  and  then  commences  to  decline ;  hence  it  is 


THE    PHENOMENA   OF   ANTITOXIN    FORMATION  65 

advisable  that  the  animal  should  be  bled  for  antitoxin  after  a  rest 
of  about  a  week  from  its  last  injection. 

The  bleedings  are  carried  out  at  the  laboratories  of  the  Royal 
Colleges  of  Physicians  and  Surgeons  in  the  following  manner  : 
The  receptacles  for  the  blood  are  2-pound  glass  jam-jars,  which 
are  sterilized  by  heat  and  covered  with  parchment  paper  which 
has  been  soaked  for  some  hours  in  i  :  20  carbolic.  Two  layers 
of  this  are  used,  and  the  lower  one  has  two  radial  slits  cut  in  it, 
leaving  a  triangular  wedge,  which  can  be  raised  and  access  to  the 
bottle  thus  obtained.  Twelve  or  fourteen  of  these  are  required 
for  each  horse,  and  each  is  filled  about  two-thirds  full. 

The  side  of  the  horse's  neck  is  shaved  and  washed  with  a 
solution  of  lysol,  or  a  lysol  dressing  is  put  on  an  hour  or  two 
before  the  operation.  The  horse  is  placed  in  the  stocks,  and  if 
violent  the  head  is  restrained  by  a  twitch.  It  is  then  necessary 
to  apply  pressure  at  the  lower  part  of  the  neck,  in  order  to  distend 
the  jugular  vein  ;  this  may  be  done  by  the  thumb  of  an  assistant, 
or,  better,  by  means  of  a  firm  leather  plug,  which  is  pressed  into 
the  groove  in  front  of  the  sterno-mastoid  muscle  by  means  of  an 
arrangement  of  straps  devised  by  Dr.  Cartwright  Wood.  In  this 
way  the  vein  is  temporarily  occluded,  and  stands  out  clearly 
above  the  region  where  the  pressure  is  applied.  The  operator 
(having  sterilized  his  hands  as  for  a  surgical  operation)  then 
makes  an  incision  about  2  inches  long  and  above  or  just  in- 
ternal to  the  vessel ;  this  should  open  the  deep  fascia,  but 
need  not  actually  expose  the  vein.  He  then  takes  a  trocar  and 
cannula  having  a  diameter  of  about  T\  inch,  and  pushes  it  firmly 
downwards  into  the  vein  ;  success  in  this  is  shown  by  the  blood 
oozing  up  by  the  side  of  the  trocar.  An  assistant  now  stands 
ready  with  a  short  metal  tube  which  fits  inside  the  cannula 
and  communicates  with  2  or  3  yards  of  indiarubber  tubing,  with 
a  foot  or  so  of  glass  tubing  at  its  farther  end.  The  whole  has 
been  sterilized  by  being  soaked  in  lysol  or  carbolic  lotion.  A 
second  assistant  now  reflects  half  of  the  outer  parchment  covers 
of  one  of  the  jam-pots,  reflects  the  triangular  strip  which  has'been 
already  cut  in  the  inner  cover,  and  inserts  the  glass  tube  in  the 
opening.  The  operator  then  removes  the  trocar,  and  the  first 
assistant  rapidly  fits  the  metal  tube  attached  to  the  rubber  tubing 
into  the  cannula;  when  this  is  done  quickly  hardly  any  blood 
escapes.  The  blood  now  passes  through  the  rubber  tubing  into 
the  jam-pot,  which  rapidly  fills.  When  about  two-thirds  full  the 

5 


66         PREPARATION    OF   ANTITOXIN    ON    A   LARGE    SCALE 

assistant  pinches  the  indiarubber  tube  and  places  the  outflow  tube 
in  a  second  pot.  The  outer  cover  is  replaced  on  the  first  pot, 
which  is  removed  to  a  warm  place  to  clot.  The  process  is  repeated 
until  twelve  or  fourteen  pots  have  been  filled. 

Horse's  blood  coagulates  slowly,  and  a  well-marked  buffy  coat  is 
formed.  In  twenty-four  hours  this  will  have  contracted,  and 
much  of  the  serum  will  be  squeezed  out.  In  order  to  draw  this 
off  use  is  made  of  a  wash -bottle,  the  short  tube  of  which  is  con- 
nected with  a  water-pump,  such  as  is  used  for  filters,  by  which 
a  partial  vacuum  can  be  maintained.  The  long  tube  is  connected 
to  a  piece  of  indiarubber  tubing  terminating  in  a  length  of  glass 
tubing.  A  jam-pot  is  opened  by  half  reflecting  the  outer  cover 
and  lifting  the  triangular  strip  cut  in  the  inner  one,  and  the  glass 
tube  is  inserted.  Air  is  now  sucked  out  of  the  wash-bottle  by 
turning  the  tap  which  puts  it  into  communication  with  the  suction- 
pump,  and  the  serum  siphons  over.  When  all  the  serum  has  been 
abstracted  a  second  jar  is  treated  in  the  same  way,  the  parchment 
cover  of  the  first  being  replaced,  and  the  process  is  continued  with 
all  the  jars.  In  twenty-four  hours  more  serum  will  have  appeared, 
and  the  process  is  repeated,  and  a  small  amount  may  often  be 
obtained  on  the  third  day.  In  this  way  the  total  yield  of  anti- 
toxin is  usually  nearly  50  per  cent,  of  the  total  volume  of  blood 
(4i  to  5  litres). 

The  antitoxin  thus  obtained  is  usually  sterile,  the  most  careful 
precautions  being  taken  to  prevent  contamination.  Carbolic  acid 
(0-3  per  cent.),  or  trikresol  (0-3  per  cent.),  or  a  mixture  of  the  two, 
must  now  be  added  to  preserve  it.  It  is  then  filtered  through 
a  Berkefeld  filter  (not  a  Chamberland  filter,  through  which  it 
passes  with  great  difficulty,  if  at  all),  a  low  pressure  only  being 
used,  and  finally  tested  for  sterility  by  means  of  cultures,  and  for 
the  presence  of  toxins  by  the  injection  of  large  (10  c.c.  or  more) 
amounts  into  normal  guinea-pigs. 

A  specimen  is  taken  at  the  time  of  the  bleeding,  and  this  is 
tested  for  antitoxic  value  in  the  manner  to  be  described  subse- 
quently. The  results  of  this  testing  will  give  the  amount  necessary 
to  obtain  the  required  dose,  and  this  amount  is  placed  in  sterile 
tubes  or  bottles  ready  for  use.  In  most  cases  mixtures  are  made, 
antitoxin  of  low  potency  being  mixed  with  more  powerful  sera  in 
order  to  obtain  the  requisite  dose  in  a  given  volume  of  serum. 
An  ingenious  machine  is  used  by  which  the  tubes  or  bottles  are 
filled  automatically  with  the  antitoxin  in  the  required  amounts. 


THE    PHENOMENA   OF   ANTITOXIN    FORMATION  67 

In  the  earlier  stages  of  immunization,  as  we  have  seen,  each 
injection  is  followed  (after  a  negative  phase)  by  a  rise  in  the  anti- 
toxic value  of  the  serum  above  its  previous  level.  In  the  stage 
which  now  follows  this  does  not  occur,  or  not  definitely  ;  there  is 
a  negative  phase,  but  it  is  found  impossible  to  force  the  antitoxic 
value  above  a  certain  level,  which  varies  in  different  horses. 
This  second  stage,  or  period  of  maintained  maximum,  varies  in 
different  horses,  and  may  last  a  few  months  or  a  year.  While  it 
lasts  there  are,  of  course,  oscillations  ;  it  falls,  for  instance,  if  the 
animal  contracts  any  disease  or  suffers  in  general  health,  but  its 
general  average  is  about  the  same. 

Sooner  or  later  this  state  of  affairs  changes,  and  the  antitoxic 
value  of  the  serum  begins  to  fall,  and  cannot  be  raised  or  even 


FIG.  7. 

a,  b,  Normal  resisting  power ;  b,  c,  period  of  hypersensitiveness ;  c,  d,  period 
of  rise  in  immunity ;  and  d,  e,  maintained  high  level  thereof.  /,  g,  Normal 
amount  of  antitoxin  ;  g,  h,  period  in  which  it  increases  ;  and  h,  i,  gradual 
fall  and  ultimate  (theoretical)  disappearance. 

maintained  at  its  former  level  in  spite  of  very  large  doses  of  toxin. 
It  trends  steadily  downward,  although  the  animal  may  continue 
to  give  useful  serum  for  a  long  time.  Thus  in  one  of  Atkinson's 
best  horses  (out  of  a  series  of  100)  the  serum  contained  1,000  to 
1,100  antitoxic  units  per  cubic  centimetre  for  ten  months,  and 
then  gradually  sank,  but  remained  above  300  units  per  cubic 
centimetre  for  two  years. 

After  a  prolonged  rest  the  power  of  manufacturing  antitoxin 
may  return,  but  then  only  lasts  a  short  time,  and  cannot  be  re- 
newed again. 

The  third  stage,  therefore,  consists  in  a  gradual  disappearance 
of  antitoxin  from  the  blood,  without  any  loss  of  the  immunity  to  the 
toxin.  It  would  seem,  indeed,  as  if  the  immunity  reaches  its 
highest  level  at  this  point,  in  spite  of  the  almost  complete  absence 

5—2 


68  IMMUNITY    NOT   DEPENDENT   ON    ANTITOXIN 

of  antitoxin.  Thus  the  two  phenomena  do  not  run  paripassu  with 
one  another.  This,  is  illustrated  in  the  foregoing  diagram,  which 
represents  in  a  purely  schematic  way  the  period  of  immunization 
and  utility  of  an  antitoxin  horse,  the  height  of  the  continuous  line 
from  the  base  representing  the  degree  of  immunity,  that  of  the 
dotted  line  the  amount  of  antitoxin  in  the  blood. 


CHAPTER  IV 

INTERREACTIONS  OF  TOXIN  AND  ANTITOXIN 

STARTING  from  the  facts  that  a  suitable  dose  of  antitoxin  will 
prevent  the  development  of  symptoms  if  toxin  is  injected  shortly 
before,  at  the  same  time,  or  shortly  after,  or  that  if  antitoxin  and 
toxin  be  mixed  in  vitro  and  injected  subsequently,  no  symptoms 
develop,  we  have  to  inquire  the  mechanism  by  which  this  is  brought 
about.  Two  theories  suggest  themselves  at  once.  The  antitoxin 
might  act  on  the  cells  of  the  living  body  in  such  a  way  as  to  render 
them  insusceptible  to  the  action  of  the  poison,  or,  in  other  words, 
render  them  immune,  or  the  toxin  and  antitoxin  might  unite 
chemically  to  form  an  inert  and  harmless  compound.  When  the 
fundamental  facts  of  antitoxic  action  were  first  discovered,  the 
majority  of  pathologists  probably  inclined  to  the  former  alternative, 
the  latter  seeming  too  simple  and  teleological.  A  certain  amount 
of  experimental  evidence  was  also  forthcoming  in  favour  of  this 
view,  but  as  this  has  a  merely  historical  value  it  will  not  be 
considered.  It  is  now  fully  proved  that  toxin  and  antitoxin  form 
chemical  compounds,  and  that  the  prophylactic  and  curative  value 
of  the  latter  is  to  be  explained  simply  on  the  grounds  that  this 
compound  is  inert,  or  devoid  of  toxic  action  on  the  animal  cells. 
The  evidence  in  favour  of  the  occurrence  of  this  chemical  com- 
bination requires  brief  discussion. 

The  first  group  of  experiments  pointing  in  this  direction  are 
those  in  which  the  toxin  and  antitoxin  are  mixed  in  vitro,  and 
the  result  tested  by  means  of  red  blood-corpuscles  as  indicators, 
the  intervention  of  the  cells  of  the  living  body  being  thus  excluded. 
(Many  of  these  experiments  can  be  repeated  on  corpuscles  which 
have  been  heated  to  a  temperature  sufficient  to  destroy  the  life  of 
isolated  body  cells,  and  the  possible  objection  that  the  corpuscles 
are  "  surviving  "  thus  removed.) 

The  first  of  these  researches  was  that  of  Ehrlich,  who  showed 

69 


70  FILTRATION    EXPERIMENTS 

that  the  agglutinative  action  of  ricin  on  red  blood-corpuscles 
could  be  inhibited  in  vitro  by  means  of  the  serum  of  an  immunized 
animal.  Kanthack  showed  that  the  action  of  snake -venom  in 
inhibiting  the  coagulation  of  blood  was  similarly  prevented  in 
vitro  by  its  appropriate  serum,  whilst  Kossel  and  others  did  the 
same  for  the  haemoglobin  of  eel's  blood,  and  Ehrlich  for 
tetanolysin.  The  previous  cases  were  not  of  true  bacterial  toxins, 
and  might  possibly  be  open  to  objection  on  that  account.  The 
experiment  of  Neisser  and  Wechsberg  on  the  effect  of  leucocidin 
on  leucocytes  in  vitro,  and  its  inhibition  by  means  of  an  antiserum, 
is  another  case  in  point.  It  is  true  that  in  this  case  the  leucocytes 
are  living,  but  we  can  hardly  imagine  that  they  have  become 
immunized  by  the  action  of  the  serum,  or  that  the  phenomenon 
can  be  explained  on  any  hypothesis  other  than  that  the  toxin  and 
its  antiserum  have  combined. 

The  second  and  most  important  series  of  researches  are  those 
of  Martin  and  Cherry,  who  show  that  several  toxins  (e.g.,  that 
of  diphtheria  and  snake-venom)  pass  through  a  porcelain  filter 
which  is  impregnated  with  gelatin,  whereas  their  appropriate 
antitoxins,  being  composed  of  larger  molecules,  do  not.  (This  had 
previously  been  proved  by  Brodie.)  They  found,  further,  that 
when  a  mixture  of  toxin  and  antitoxin  was  placed  on  such  a  filter 
the  first  portion  of  the  filtrate  was  toxic,  but  that  the  amount 
diminished,  and  all  toxicity  disappeared  a  few  minutes  after  the 
mixture  had  been  made.  The  inference  is  clear :  the  toxin  had 
united  with  the  antitoxin  to  form  a  molecule  as  large  as,  or  even 
larger  than,  that  of  the  latter,  and  therefore,  like  it,  unable  to  pass 
through  the  pores  of  the  filter.  These  researches  have  been 
confirmed  by  Brodie,  and  form,  on  the  whole,  the  most  striking 
direct  proof  of  the  union  of  the  two  substances  yet  brought 
forward. 

Calmette  found  that  snake-venom  is  more  heat-resistant  than 
its  antitoxin,  withstanding  a  temperature  of  80°  or  90°  C.,  whereas 
the  latter  is  rendered  inert  at  68°  C.  He  was  then  able  to  show 
that  a  neutral  mixture  of  the  two  could  be  rendered  toxic  again 
by  exposure  to  a  temperature  of  70°  C. ;  and  this  fact  was  used  first 
as  an  argument  against  the  chemical  theory  of  combination,  and 
secondly  as  a  proof  that  the  toxin  is  not  destroyed  when  it  unites 
with  antitoxin.  As  a  matter  of  fact,  neither  inference  is 
necessarily  correct,  and  the  experiment  was  shown  by  the  further 
researches  of  Martin  and  Cherry  to  constitute  a  proof  of  the 


INTERREACTIONS   OF   TOXIN    AND   ANTITOXIN  71 

chemical  theory :  for  they  found  that  if  the  mixture  were  allowed 
to  stand  for  some  time  at  the  temperature  of  the  body  before  being 
heated,  its  toxicity  was  not  restored  by  a  temperature  of  70°  C. 
This  seems  to  show  that  the  toxin  did  not  exist  as  such  in  the 
mixture,  otherwise  it  would  not  have  been  destroyed  by  the  heat ; 
it  must,  therefore,  have  become  combined  with  the  antitoxin,  or 
at  any  rate  modified  by  it  in  some  way.  On  the  other  hand,  the 
experiment  does  not  prove  that  the  toxin  is  completely  destroyed 
beyond  all  power  of  further  activity ;  it  simply  shows  that,  when 
in  a  condition  of  combination  with  its  antitoxin,  it  is  less  thermo- 
stable than  when  free.  Similar  facts  were  adduced  by  Wasser- 
mann  with  regard  to  the  combination  between  pyocyaneus  toxin 
and  its  antitoxin,  and  are  capable  of  a  similar  explanation. 
Marenghi  has  also  brought  forward  somewhat  similar  results 
with  diphtheria  toxin. 

Lastly,  Ehrlich  has  shown  that  the  conditions  which  favour  the 
occurrence  of  chemical  combinations  favour  the  union  of  toxin 
and  antitoxin — e.g.,  it  is  accelerated  by  heat,  and  takes  place  more 
quickly  in  concentrated  than  in  dilute  solutions. 

This  brings  us  to  the  question  as  to  whether  the  combination 
takes  place  in  accordance  with  the  law  of  multiple  proportions — 
a  question  of  great  difficulty,  but  one  which  has  lead  in  its 
elucidation  to  the  discovery  of  facts  of  much  interest.  As  far  as 
concerns  the  action  of  the  haemolysins  and  other  toxins  that  can 
be  readily  tested  in  vitro,  there  is  no  doubt  that  this  question,  in 
its  simplest  form,  must  be  answered  in  the  affirmative.  If  it 
requires  x  c.c.  of  a  given  solution  of  toxin  to  dissolve  exactly 
i  c.c.  of  a  5  per  cent,  emulsion  of  red  blood-corpuscles,  then  it 
will  require  2#,  3*,  4*,  etc.,  c.c.  to  haemolyze  2,  3,  4,  etc.,  c.c.  of 
the  same  emulsion.  We  assume  in  each  case  that  the  haemolysin 
is  added  at  once,  and  not  in  small  consecutive  amounts.  To 
study  the  effect  of  the  partial  neutralization  of  toxin  by  antitoxin 
we  will  briefly  outline  Ehrlich's  famous  work  on  the  standardiza- 
tion of  diphtheria  toxin,  and  the  conclusions  he  arrived  at  in 
consequence  of  the  results  thus  obtained. 

We  have  seen  that  it  is  possible  to  determine  with  a  close 
approach  to  accuracy  the  minimal  lethal  dose  of  diphtheria  toxin 
for  standard  guinea-pigs— 4.e.,  those  weighing  about  250  grammes. 
This  amount  is  called  the  toxic  unit  (TU),  and  a  toxin  of  which 
T^  c.c.  is  just  sufficient  to  kill  a  test  guinea-pig  in  three  or  four 
days  is  considered  to  be  normal  toxin  of  unit  strength,  and  is 


72  STANDARDIZATION    OF    DIPHTHERIA   TOXIN 

written  DTN.1  A  toxin  of  half  this  strength,  of  which  -^  c.c. 
is  the  lethal  dose,  is  written  DTN0.5.  Toxins  of  other  potencies 
are  numbered  accordingly. 

Ehrlich  now  proceeded  to  define  a  unit  of  antitoxin  as  the 
amount  that  would  just  neutralize  100  lethal  doses  of  toxin  :  this 
is  called  IU  (  =  immunizing  unit).  This  amount  may  be  contained 
in  any  quantity  of  the  serum ;  thus,  in  that  used  for  clinical  work 
i  c.c.  contains  anything  between  300  and  1,000  units,  or  even 
more.  For  the  purpose  of  testing  toxins  it  is  convenient  to  use 
an  antitoxic  serum  which  is  much  more  dilute  than  this,  and  an 
antitoxin  of  unit  strength  is  defined  as  one  which  contains  i  unit 
of  antitoxin  in  i  c.c. — i.e.,  i  c.c.  of  the  antitoxin  will  just  neutra- 
lize i  c.c.  (100  lethal  doses)  of  standard  toxin.  The  reaction 
between  these  amounts  is  written  thus  : 

i  c.c.  toxin  (=100  lethal  doses)  +  i  c.c.  antitoxin  =  L0, 

where  L0  (L  =  limes)  indicates  that  the  mixture  is  a  truly  neutral 
one,  and  that  it  does  not  kill  a  susceptible  animal  within  the  time- 
limit,  or  produce  any  pathogenic  action  whatever. 

Now,  if,  as  Ehrlich  believes,  the  affinity  of  toxin  for  antitoxin 
is  a  powerful  one,  similar  to  that  of  a  strong  acid  for  a  strong 
base,  it  should  follow  that  if  to  the  100  lethal  doses  of  toxin  we 
add  only  T9^  of  i  c.c.  of  standard  antitoxin,  then  TJy  of  the  original 
amount — i.e.,  i  lethal  dose — should  remain  unneutralized,  and  the 
animal  should  die  in  the  same  time  as  a  similar  animal  which  had 
received  i  lethal  dose  and  no  antitoxin. 

As  a  matter  of  fact,  this  is  not  what  occurs.  We  find  that  when 
we  inject  the  mixture  the  animal  does  not  die  in  a  short  time 
with  the  ordinary  symptoms  of  diphtheritic  intoxication,  but 
develops  local  oedema,  and  possibly  paralysis,  which  may  bring 
about  death  at  a  remote  period.  The  same  thing  happens  if  we 
add  still  less  antitoxin  to  the  100  lethal  doses  of  toxin.  To  take 
a  particular  case,  it  is  not  until  the  mixture  contains  less  than 
T7^y  of  i  c.c.  of  antitoxin  that  the  animal  dies  acutely  in  the  way 
it  does  after  an  injection  of  i  lethal  dose  of  toxin.  It  seems, 
therefore,  that  the  whole  of  the  toxicity  of  the  toxin  is  removed 
when  only  three-fourths  of  the  amount  of  antitoxin  necessary  to 
neutralize  it  has  been  added,  or  that  a  given  amount  of  toxin  can 

1  DTN  =  diphtheria  toxin  normal.  It  is  also  written  DTN1M250=DTN  one 
unit  for  a  guinea-pig  (Meerschweinchen)  weighing  250  grammes. 


INTERREACTIONS   OF   TOXIN    AND   ANTITOXIN 


73 


combine  with  one -fourth  more  antitoxin  than    is   necessary  to 
neutralize  it. 

To  account  for  this  Ehrlich  supposed  that  there  are  really  two 
substances  present  in  the  broth  in  which  diphtheria  bacilli  have 
been  grown.  There  is  the  true  toxin,  which  brings  about  local 
inflammatory  oedema,  often  going  on  to  necrosis  and  causing  local 
alopecia,  and  causing  acute  death,  and  toxon,  which  produces  only 
soft  and  transient  cedema  locally  and  subsequent  paralysis.  Both 
these  substances  combine  with  antitoxin,  but  the  toxin  has  the 
greater  affinity  for  that  substance,  and  when  the  total  neutralizing 
dose  of  antitoxin  is  added  in  successive  small  amounts,  the  whole 
of  the  toxin  is  neutralized  first,  leaving  the  toxon  free,  and  this 
takes  place  when  three-fourths  of  the  whole  amount  of  antitoxin 
has  been  added.  Ehrlich  represents  this  result  in  the  form  of  a 
spectrum,  thus : 


0  25  50  75          100 

FIG.  8.— SIMPLE  SPECTRUM  OF  TOXIN. 

The  rectangle  represents  the  L0  dose  of  toxin — i.e.,  in  this  simple 
case  i  c.c.  of  the  solution.  The  portion  with  the  greatest  affinity 
for  antitoxin  is  placed  at  the  left  hand  of  the  "spectrum";  in 
this  case  it  is  represented  by  the  toxin.  On  the  right  are  the 
substances  with  the  least  affinity  for  antitoxin — in  this  case  the 
toxon. 

Further  investigation  shows  that  the  process  is  not  usually  so 
simple  as  this.  In  certain  samples  of  toxin  we  find  that  the 
addition  of  small  quantities  of  antitoxin  causes  no  alteration  in  the 
toxicity  of  the  L0  dose.  Thus,  in  a  case  of  frequent  occurrence 
it  happens  that  we  may  add  J  c.c.  of  normal  antitoxin  before 
any  loss  of  toxicity  occurs  ;  i  c.c.  of  the  normal  toxin  will  kill 
100  guinea-pigs,  and  i  c.c.  of  the  same  toxin  +  J-  c.c.  of  normal 
antitoxin  will  still  kill  100  guinea-pigs.  To  explain  this,  Ehrlich 
supposed  that  the  solution  contains  a  third  substance,  prototoxoid, 
which  is  entirely  devoid  of  lethal  activity,  but  which  has  a  power 
of  combining  with  antitoxin  even  greater  than  that  which  toxin 
possesses.  Thus,  on  the  addition  of  small  amounts  (up  to  J  c.c.)  of 
the  antitoxin,  this  inert  substance  will  seize  on  the  antibody,  unite 


74 


STANDARDIZATION    OF    DIPHTHERIA    TOXIN 


with  it,  and  so  render  it  incapable  of  neutralizing  the  true  toxin. 
The  spectrum  of  this  solution  will  be  represented  thus  : 


Pntto- 
toxoid 


Toxone 


FIG.  9. — SPECTRUM  OF  TOXIN. 

In  this,  as  in  the  other  diagrams,  the  lethal  portion  of  the  mixture 
is  shaded,  the  non-lethal  portion  left  blank. 

Ehrlich  found  on  actual  experiment  that  the  constitution  of  the 
solution  was  even  more  complex  than  this,  and  had  to  assume  the 
existence  of  yet  other  bodies.  Thus,  if  the  spectrum  above  were 
a  true  representation  of  the  constitution  of  i  c.c.  of  the  solution,  it 
follows  that  the  first  quarter  and  the  last  quarter  of  the  antitoxin 
added  were  without  effect,  so  that  the  middle  \  c.c.  completely 
neutralized  the  whole  of  the  TOO  lethal  doses.  Now  let  us 
imagine  this  i  c.c.  of  standard  antitoxin  divided  into  200  equal 
parts,  and  added  part  by  part  to  the  i  c.c.  of  standard  toxin,  or 
100  lethal  doses.  Then — 

The  first  50  parts  added  will  combine  with  prototoxoid,  and  will 

not  affect  the  toxicity  of  the  mixture ; 
The  next  100  parts  added  will  neutralize  100  lethal  amounts 

of  true  toxin  ;  and 
The  last  50  parts  will  combine  with  toxon. 

Now  if  the  spectrum  were  as  simple  as  is  shown  above,  and  if 
the  toxin  were  quite  uniform  in  its  combining  capacity  and  its 
toxicity,  it  would  follow  that  the  first  -^^  part  added  after  the 
addition  of  ^^  part  would  just  neutralize  one  lethal  dose  and  leave 
99  lethal  doses  over.  Again,  the  addition  of  the  amount  necessary 
to  neutralize  all  the  prototoxoid  (  =  -f^  c.c.)  +  -££$  c.c.,  which  would 
neutralize  all  the  prototoxoid  and  all  the  toxin  except  yj^  part 
(=i  lethal  dose),  and  all  the  toxon,  should  leave  i  lethal  dose 
of  toxin  free,  and  the  animal  should  die  in  the  limit  of  time  for 
i  lethal  dose.  We  might  represent  this  as  follows : 

149     unnc'jfralised 
'Toxin  ( 


INTERREACTIONS   OF   TOXIN    AND   ANTITOXIN  75 

in  which  the  oblique  shading  represents  the  toxic  portions,  as 
before,  and  the  horizontal  shading  represents  the  amount  neutral- 
ized by  the  addition  of  ^-^  c.c.  of  antitoxin ;  the  portion  with 
oblique  but  no  horizontal  shading  represents  the  toxic  portion 
which  remains  unneutralized :  it  constitutes  TJ^  of  the  total 
shaded  portion,  and  is  therefore  i  lethal  dose. 

Such  a  finding  may  occur,  but  is  unusual.  In  most  cases  we 
find  that  the  amount  of  toxin  left  free  on  partial  neutralization  is 
subject  to  laws  which  are  far  more  complex.  In  a  case  given  by 
Madsen  and  described  in  the  same  way  we  find  : 

The  addition  of  |££  parts  of  antitoxin  left  free  no  lethal  substance 
— a  term  which  we  shall  use  for  the  present,  instead  of  "  toxin," 
to  denote  the  portion  of  the  spectrum  with  the  oblique  shading. 
In  Ehrlich's  language  all  had  been  neutralized  except  the  toxon. 

The  addition  of  ££$  left  5  units  of  lethal  substance  free ;  it 
follows  that  i§S~  29^j-=2fio°i7  had  been  necessary  to  neutralize 
these  5  units. 

The  addition  of  -f^  left  55  lethal  units  free  ;  hence,  if  after 
the  addition  of  -f^  (as  above,  leaving  5  lethal  doses  free)  we  add 
an  additional  -/^,  the  difference  (•/<&)  will  neutralize  50  lethal 
doses  (55-5). 

Hence  the  additon  of  •££§  will  just  neutralize  the  remaining 
lethal  doses — i.e.,  45. 

To  account  for  facts  like  these,  Ehrlich  suggests  that  the 
solution  contains  four  or  five  substances.  The  first — i.e.,  that 
which  has  the  greatest  power  of  combining  with  antitoxin,  is  called 
prototoxin ;  it  is  lethal,  and  it  consists  of  two  parts — an  a  part, 
which  is  readily  changed  into  inert  prototoxoid,  and  a  ft  part, 
which  is  more  stable,  but  which  may,  after  a  time,  change  into 
prototoxoid  also.  These  two  modifications  have  exactly  the  same 
affinity  for  antitoxin,  so  that  if  they  were  present  in  equal 
amounts,  and  if  all  the  a  modification  were  changed  into  proto- 
toxoid, each  addition  of  antitoxin  would  go  to  neutralize  active 
prototoxin  and  inert  prototoxoid  in  equal  amount ;  hence  half  of 
it  would  apparently  be  wasted. 

Secondly,  there  is  deuterotoxin,  which  also  exists  in  an  a  and  a  /? 
modification,  of  which  the  a  part  is  readily  transformed  into 
deuterotoxoid,  whilst  the  (3  modification  is  very  stable  and  is  the 
last  lethal  substance  to  disappear.  The  a  and  /3  modifications 
have  equal  affinity  for  antitoxin,  but  this  is  less  than  that  of  the 
prototoxin. 


76 


SPECTRA   OF   TOXINS 


Thirdly,  there  is  tvitotoxin,  again  in  an  a  and  a  /3  modification, 
with  less  affinity  for  antitoxin  than  deuterotoxin,  and  so  are  placed 
on  its  right  in  the  spectrum. 

It  is  found,  further,  that  the  proportion  of  a  modification  to 
P  modification  in  the  above  forms  of  toxin  is  a  simple  one, 
so  that  the  ratio  of  toxoid  to  toxin  present  in  any  one  part  of  the 
spectrum  is  always  simple  (-J,  ^,  ^,  etc.). 

Fourthly,  there  is  toxon  (toxone)  or  epitoxoid,  the  characters  of 
which  we  have  seen. 

Lastly,  some  researches  seem  to  prove  that  there  is  yet 
another  body,  epitoxonoid,  which  has  still  less  affinity  for  antitoxin 
than  has  toxon,  and  which  is  entirely  devoid  of  lethal  or  toxic 
power.  It  will  be  left  out  of  the  further  consideration  of  these 
bodies. 

The  spectrum  of  a  toxin  on  this  theory  is  recorded  thus : 


Profofaxo/clA 


Trifofoxoicf  A 


Toxon 


Profotoxin  B      Deuterotoxin  B  Tritotoxin  B 

FIG.  ii. 


The  spectrum  of  the  example  given  by  Madsen  and  quoted 
above  would  be  : 


FIG.  12. 
Another  spectrum,  given  by  Ehrlich,  is  appended  : 


DeuferotoxinB  Tritoxoxm  B 

FIG.  13. 


We  must  now  turn  to  the  experimental  results  which  have  led 
to  this  idea  of  the  change  of  the  toxin  into  toxoid ;  it  has  been 


INTERREACTIONS    OF   TOXIN    AND    ANTITOXIN  77 

referred  to  several  times  already,  but  not  fully  discussed  in  order 
not  to  interrupt  the  main  line  of  the  argument. 

L0  has  been  denned  as  the  amount  of  toxic  solution  which  is 
exactly  neutralized  by  i  IU  of  antitoxin,  and  L+  is  the  amount 
which,  when  added  to  i  IU  of  antitoxin  has  i  lethal  dose  left  un- 
neutralized.  Now  if  the  toxic  solution  contained  a  simple  sub- 
stance, we  should  expect  the  two  quantities  to  have  the  following 
relation  in  the  simple  standard  toxin  of  which  i  c.c.  contains  100 
lethal  doses. 

i  c.c.  toxin(=  100  lethal  doses)  +  i  c.c.  antitoxin  (=  i  IU)  =  L0. 
roi  c.c.  toxin(=  101  lethal  doses)  +  i  c.c.  antitoxin  ( =  i  IU)  =  L+. 
.-.  L+-L0  =  o-oi  c.c.  =  i  lethal  dose. 

This,  however,  is  not  the  case.  If  we  take  a  neutral  mixture  of 
toxin  and  antitoxin — e.g.,  of  100  units  of  the  former  and  i  of  the 
latter — add  to  it  i  lethal  dose  of  toxin,  and  inject  it  into  an  animal, 
it  will  not  cause  death  ;  there  may  be  transient  local  cedema  and 
late  paralysis,  symptoms  which  are  indicative  of  the  presence  of 
free  toxon.  We  must  in  general  add  very  much  more  than 
i  lethal  dose  to  the  neutral  mixture  in  order  to  bring  about  a  fatal 
result.  For  example,  in  our  standard  toxin  it  might  happen  that 
the  L+  dose  was  about  1-35  c.c.  In  other  words— 

i -oo  c.c.  toxin  solution  +  i  unit  of  antitoxin  =  L0. 
I*35  c.c.  toxin  solution  +  i  unit  of  antitoxin  =  L+. 
L+-L0  =  o-35  c.c. 

This  result  can  readily  be  explained  on  Ehrlich's  assumption 
of  the  existence  of  substances  of  differing  combining  powers  for 
antitoxin.  For  the  sake  of  simplicity,  we  will  take  his  earlier 
nomenclature,  and  consider  the  substance  as  made  up  of  proto- 
toxoid  (with  a  greater  affinity  for  antitoxin  than  true  toxin  has), 
toxin,  and  epitoxoid,  with  little  affinity,  and  corresponding  to 
toxon.  The  spectrum  of  the  toxin  under  discussion  is : 


150  200 

FIG.  14. 


In  this  diagram  we  represent  the  L0  dose — i.e.,  i  c.c.  divided 
into  its   component  parts.     The   oblique   shading  represents,  as 


78 


DIFFERENCE    BETWEEN    L. 


before,.Jthe  acutely  lethal  portion,  and  the  whole  is  shaded  hori- 
zontally to  show  that  it  is  completely  neutralized  by  the  i  unit  of 
antitoxin. 

Now  let  us  take  1*25  of  the  same  solution  and  add  to  it  i  unit 
of  antitoxin.  In  this  extra  0-25  c.c.  of  toxin  (a  quarter  of 
the  original  amount)  there  are  12-5  parts  of  prototoxoid,  25  of 
toxin,  and  12-5  of  epitoxoid.  There  will  now  be  62-5  parts  of 
prototoxoid,  125  of  toxin,  and  62-5  parts  of  epitoxoid.  The  200  parts 
into  which  we  imagine  the  unit  of  antitoxin  is  divided  will  now 
neutralize  the  whole  of  the  prototoxoid  (62-5  parts),  the  whole  of 
the  toxin  (125  parts),  and  12-5  parts  of  toxon.  There  will  be 
50  parts  of  epitoxoid  left  free,  but  no  toxin.  Hence,  1*25  c.c.  of 
the  toxic  solution  is  less  than  the  L+  dose.  The  result  may 
be  represented  thus : 

n-6  parts  of  epifoxotd 


62-5  parrs 


125  parrs. 
FIG.  15. 


200    62-5  parrs 


Let  us  now  imagine  a  third  mixture  of  1-33  c.c.  of  the  toxic 
solution  and  i  unit  of  antitoxin.  The  0-33  c.c.  of  toxin  will 
contain  16-6  c.c.  of  prototoxoid,  33-3  c.c.  of  toxin,  and  16*6  c.c.  of 
toxon,  and  the  total  1*33  c.c.  will  thus  contain  66-6  c.c.  of  proto- 
toxoid, i33'3  c.c.  of  toxin,  and  66*6  c.c.  of  epitoxoid.  The  proto- 
toxoid +  toxin  (  =  200  parts)  will  just  absorb  the  whole  of  the  unit 
of  antitoxin,  leaving  nothing  but  toxon  free.  Thus  : 


66  6  parrs 


133-3  parrs 

FIG.  1 6. 


ZOO        66-  6  parrs 


Then,  if  i  extra  lethal  dose  of  toxin  be  added  to  the  above 
mixture,  it  will  find  all  the  antitoxin  utilized  by  substances  with 
a  combining  affinity  as  great  as,  or  greater  than,  its  own,  and 
will  be  left  free.  Hence,  the  L+  dose  is  just  greater  than 
1 33  c.c. 

All  this  follows  from  what  has  previously  been  said  concerning 


INTERREACTIONS    OF   TOXIN   AND   ANTITOXIN 


79 


partial  neutralization.  If,  however,  we  now  keep  this  atrtitoxin 
for  some  time,  especially  if  it  is  exposed  to  warmth,  light,  air, 
or  certain  chemical  substances,  we  find  a  great  change.  The 
L0  dose  is  unaltered :  i  c.c.  is  still  exactly  neutralized  by  i  unit 
of  antitoxin,  but  we  find  that  this  amount  is  now  much  less  lethal, 


Prototoxoid 


Epitoxoid 


50  parrs 


loo  parrs 
FIG.  17. 


SO  parrs 


and  the  minimal  lethal  dose  may  have  risen  from  o'oi  c.c.  to 
O'O2  c.c.,  or  higher. 

If  the  cause  for  this  increase  in  the  lethal  doses  is  investigated 
by  the  partial  neutralization  method  described  above,  it  will  be 
found  that  the  results  obtained  are  such  as  will  be  readily  ex- 
plicable on  the  assumption  that  some  of  the  molecules  of  toxin 


Profotoxoid. 


Toxoid  (SO  parts) 


Epiroxoid 


50  parts 


100  parts 
FIG.  18. 


50  parrs 


have  ceased  to  be  poisonous,  but  have  retained  their  combining 
power  unaltered;  whilst  the  non  -  poisonous  portions  of  the 
spectrum  are  unaltered.  Thus,  to  take  the  simple  case  described 
above,  and  shown  in  Fig.  17,  in  which  protoxoid,  toxin,  and 
epitoxoid  are  present  in  the  proportion  of  50,  100,  and  50.  If  we 
keep  this,  we  may  find  the  lethal  dose  doubled — i.e.,  ^  c.c.  instead 
of  T<JTT  c-c-  But  on  working  out  the  action  of  antitoxin,  we  may 
find  that  the  first  -25  c.c.  added  removes  none  of  the  toxicity,  and 
the  last  -25  is  equally  without  apparent  effect.  Thus  the  middle 
•5  c.c.  is  used  up  in  neutralizing  50  lethal  doses,  and  of  this 
fraction  further  investigation  shows  that  each  one-fiftieth  part 
neutralizes  one  lethal  dose;  the  antitoxin,  therefore,  appears  to 
have  fallen  off  in  potency ;  but  we  know  that  this  is  not  the  case. 
The  explanation  is  that  half  the  molecules  have  been  changed  into 
the  non-toxic  form  described  above.  The  spectrum  of  this  altered 
form  is  shown  in  Fig.  18. 

Thus,  in  a  particular  case  Ehrlich  found  the  minimal    lethal 


80  TOXIN   AND   TOXOID 

dose  of  a  toxin  to  be  0*003,  and  the  L0  dose  0-305  (=  100  lethal 
doses).  Nine  months  later  the  L0  dose  was  still  0305,  whilst 
the  minimal  lethal  dose  was  0-009  c-c-  (=33*3  lethal  doses  only). 
Thus,  the  toxin  had  fallen  to  one-third  of  its  former  toxicity,  but 
had  retained  its  power  of  combining  with  antitoxin  unaltered  ;  and 
it  is  found  that  the  two  numbers  usually  bear  some  simple  ratio 
to  one  another — e.g.,  i  -  ^  or  i  —  J,  as  in  the  previous  examples. 

We  are  now  in  a  position  to  understand  all  Ehrlich's  results, 
and  especially  the  proof  that  a  molecule  of  toxin  consists  of  two 
parts — a  haptophore  group,  which  has  the  power  of  combining 
with  antitoxin  or  with  the  protoplasm  of  the  body  cells,  but  which 
has  in  itself  no  lethal  action ;  and  a  toxophore  group,  which  has 
the  power,  when  linked  to  a  body  cell  by  means  of  the  haptophore 
group,  of  causing  toxic  symptoms,  probably  by  a  process  akin  to 
enzyme  action.  This  toxophore  group  is  unstable,  and  when  it  is 
decomposed  the  toxin  molecule  is  converted  into  toxoid ;  it  then 
retains  its  power  of  uniting  with  antitoxin  and  with  the  tissue 
cells,  but  is  devoid  of  toxicity.  Further,  there  are  many  varieties 
of  toxin,  which  differ  from  one  another — (i)  in  their  avidity  for 
antitoxin,  and  presumably  for  the  tissue  cells ;  (2)  in  the  readiness 
with  which  they  are  decomposed  into  inert  toxoid;  and  (3)  in 
their  toxic  action,  in  that  true  toxins  cause  rapid  death,  local 
inflammation,  necrosis,  etc.,  but  no  paralysis,  whilst  toxons 
produce  only  transient  soft  oedema  and  subsequent  paralysis. 
Lastly,  Ehrlich  supposes  that  toxin  and  antitoxin  have  a  great 
affinity  for  one  another,  so  that  their  combination  resembles  that 
of  a  strong  acid  with  a  strong  base,  and  the  compound  toxin- 
antitoxin  molecule  when  once  formed  does  not  dissociate,  but 
remains  as  a  stable  substance.  It  is  on  this  point  that  his  views 
differ  from  the  more  recent  ones  of  Arrhenius  and  Madsen,  who 
regard  the  union  as  being  akin  to  that  of  a  weak  acid  and  a  weak 
base.  We  hope  to  be  pardoned  if,  before  describing  their  experi- 
ments and  conclusions,  we  give  a  brief  outline  of  the  difference 
between  the  two  reactions. 

According  to  modern  chemical  theories,  most  substances  in 
watery  solution  decompose  into  ions,  atoms  or  groups  of  atoms 
carrying  an  electrical  charge.  Thus,  a  solution  of  hydrochloric 
acid  contains  ions  of  H  carrying  a  positive  charge  and  of  Cl 
carrying  a  negative  one.  Different  substances  undergo  ionization 
to  a  different  degree.  Thus,  HC1  and  NaOH  are  ionized  to  a 
great  extent,  boracic  acid  and  ammonia  hydroxide  but  slightly. 


INTERREACTIONS    OF   TOXIN    AND    ANTITOXIN  8l 

Now  in  general  it  is  only  free  ions  which  enter  into  chemical 
combination.  For  example,  on  adding  HC1  to  NaOH  the  posi- 
tively charged  H  ion  combines  with  the  negatively  charged  OH 
ion  to  form  water,  and  the  positively  charged  Na  ion  combines 
with  the  negatively  charged  Cl  ion  to  form  NaCl.  The  two 
substances  HC1  and  NaOH  are  strongly  dissociated,  and  hence 
the  combination  between  the  two  is  almost  complete.  This  is  an 
example  of  the  combination  of  a  strong  acid  and  a  strong  base ; 
where  it  was  expressed  in  older  phraseology,  the  substances  have 
a  strong  affinity  for  one  another.  It  is  to  this  type  that  Ehrlich 
conceives  the  union  of  toxin  and  antitoxin  belongs. 

When  two  substances  dissociate  but  slightly — e.g.,  acetic  acid 
and  alcohol  or  boracic  acid  and  ammonia — the  reaction  takes 
place  in  obedience  to  different  laws  (the  law  of  "mass  action" 
of  Guldberg  and  Waage).  When  we  ^dd  alcohol  and  acetic 
acid  we  get  ethyl  acetate  (ester)  and  water ;  but  if  we  take 
equivalent  combining  quantities  of  the  two  primary  substances, 
the  reaction  is  not  complete,  as  it  is  in  the  case  of  HC1  and 
NaOH.  On  the  contrary,  the  solution  will  still  contain  free 
alcohol,  free  acetic  acid,  ester,  and  water.  This  is  due  to  the 
fact  that  the  process  is  reversible.  Acetic  acid  and  alcohol 
combine  on  the  one  hand  to  form  ester  and  water,  and  ester 
and  water  combine  on  the  other,  and  dissociate  into  free  acid  and 
free  alcohol. 

In  the  first  case,  in  the  combination  of  a  strong  acid  and  a 
strong  base,  the  reaction  is  a  simple  one.  If  we  take  100  com- 
bining equivalents  of  NaOH,  and  add  to  it  10  combining  equiva- 
lents of  NaOH,  10  parts  of  the  alkali  will  be  neutralized,  and  so 
on,  the  whole  of  the  alkali  being  neutralized  when  100  combining 
equivalents  have  been  added.  In  the  second  case  the  reaction  is 
more  complicated,  and  is  expressed  by  the  law  that  the  products 
of  the  concentrations  of  the  substances  on  one  side  of  the  equa- 
tion, divided  by  the  product  of  those  on  the  other,  is  a  constant 
(which  varies  in  different  reactions,  and  can  be  determined  by 
experiment).  Thus  in  the  case  given  : 

Concentration  of  acid  x  concentration  of  alcohol 

=  constant. 
Concentration  of  ester  x  concentration  01  water 

It  follows  that  when  we  add  a  relatively  small  amount  of  acid 
to  a  given  volume  of  alcohol,  it  is  practically  all  used  up  to  form 
ester,  much  free  alcohol  remaining ;  but  each  succeeding  addition 

6 


82  REVERSIBLE    REACTIONS 

of  acid  is  divided  into  two  parts,  of  which  one  combines  with 
the  alcohol  and  the  other  remains  free.  As  we  continue  to  add 
successive  small  amounts,  this  latter  uncombined  portion  gets 
greater  and  greater,  so  that  the  addition  of  successive  small 
volumes  use  up  less  and  less  of  the  alcohol ;  and  it  is  only  when 
there  is  a  great  excess  of  acid  that  all  the  alcohol  is  used  up. 
Theoretically,  some  always  remains. 

The  difference  may  be  represented  graphically  thus  : 


FIG.  19. 

The  line  a  b  represents  the  neutralization  of  a  given  amount  of 
NaOH  by  its  equivalent  of  acid.  It  is  a  straight  line,  since  each 
successive  addition  of  equal  amounts  of  acid  neutralizes  the  same 
amount  of  alkali. 

The  line  c  d  represents  the  reaction  of  alcohol  and  acetic  acid, 
or,  to  take  the  example  used  by  Madsen,  the  neutralization  of 
ammonia  by  boracic  acid.  It  is  a  hyperbolic  curve.  It  is  almost 
a  straight  line  to  begin  with,  since  each  small  addition  of  boracic 
acid  is  almost  all  used  up  by  the  ammonia,  there  being  enough 
NH4  ions  to  seize  on  all  the  boracic  ions  which  are  added. 
Farther  along,  however,  it  changes  according  to  the  rules  already 
described,  and  ultimately  approaches  infinitely  near  to  the  base 
line,  but  never  reaches  it.  There  are  always  free  boracic  acid 
and  free  ammonia  in  the  solution,  though  the  amount  of  the  latter 
is  infinitely  small  when  the  former  is  in  great  excess. 

The  first  suggestion  that  the  reaction  between  toxin  and  anti- 
toxin might  be  a  reversible  one  was  due  to  Myers  in  an  investi- 
gation on  the  haemolytic  action  of  snake-venom,  a  substance 


INTERREACTIONS    OF    TOXIN    AND    ANTITOXIN  »3 

which  gives  partial  neutralization  phenomena  quite  similar  to 
those  we  have  described  as  occurring  with  diphtheria  toxin,  and 
also  declines  in  toxic  strength,  but  not  in  combining  capacity, 
forming  toxoids.  It  was,  however,  the  researches  of  Arrhenius 
and  Madsen  which  led  to  the  establishment  of  the  theory  on  a 
sound  basis.  Madsen  had  previously  investigated  the  constitution 
of  the  haemolysin  of  tetanus  (tetanolysin),  working  on  Ehrlich's 
lines,  and  had  found  it  to  consist  of  proto-,  deutero-,  and  trito- 
toxin,  and  a  large  amount  of  toxone.  The  substance  was  much 
more  convenient  to  work  with  than  diphtheria  toxin,  since  a  constant 
emulsion  of  red  blood-corpuscles  could  be  used  as  test  objects 
instead  of  guinea-pigs,  and  thus  the  experiment  could  be  multi- 
plied at  will ;  and  when  Madsen  added  the  antiserum  in  small 
amounts  he  found  that  the  apparent  irregularities  due  to  the 
presence  of  these  various  forms  of  toxin  disappeared,  and  the 
curve  of  neutralization  is  represented  by  a  curve  similar  to  that 
given  above,  as  illustrating  the  reactions  between  boracic  acid 
and  ammonia.  Thus : 


2     4     6     8    10    12    14    16    18  20  22  24  26  28  30 

FIG.  20. CURVE  OF  NEUTRALIZATION  OF  TETANOLYSIN  (MADSEN). 

6—2 


84 


ARRHENIUS'    AND    MADSEN's   VIEWS 


The  heavy  line  represents  the  curve  of  neutralization  on 
Ehrlich's  principles,  and  apparently  shows  the  presence  of  a 
large  number  of  varieties  of  toxin  with  different  combining 
capacities.  When,  however,  the  neutralization  was  carried  out  by 
the  addition  of  very  small  quantities  of  the  antitoxin  at  a  time, 
the  curve  became  a  hyperbola  of  the  character  seen  in  the  dia- 
gram. Further,  having  found  the  value  of  k  by  determining  by 
experiment  the  amount  of  toxin  left  free  after  the  addition  of 
a  certain  amount  of  antitoxin  to  a  certain  amount  of  the  toxic 
solution  in  two  different  instances,  and  hence,  by  use  of  the 
formula : 

Free  toxin     free  antitoxin  _     (toxin  x  antitoxin) 
vol.  vol.  vol. 

the  amount  of  free  toxin  after  any  addition  of  antitoxin  could  be 
calculated  theoretically.  It  could  then  be  determined  by  experi- 
ment, and  the  results  compared.  In  one  case  given  by  Arrhenius 
and  Madsen  this  was  done,  with  the  following  result : 


Amount  of 
Antitoxin  added. 

Amount  of  Toxin 
(observed). 

Amount  of  Toxin 
(calculated). 

0*05 

3-67 

3-67 

o-i 

3-13 

2'95 

0*15 
0-2 

2-32 
1-62 

2-29 
172 

0-3 
0-4 

07 
I'D 

0-97 
0-63 

o*45 
0-27 
0-18 

1-03 
0-62 
0-46 
0-28 
0-18 

1*3 

1-6 

0*12 

0-13 

O'll 

2-0 

0'08 

0-09 

The  correspondence  is  certainly  very  close. 

The  case  of  diphtheria  toxin  was  next  investigated,  and  in  the 
figure  which  follows  the  "  stair-step  "  curve,  showing  Ehrlich's 
conception  of  its  constitution,  shows  the  presence  of  proto-,  deutro-, 
and  trito-toxins,  and  of  toxon.  The  curved  line  is  that  calcu- 
lated after  the  constant  of  dissociation  had  been  determined  by 
experiment. 


INTERREACTIONS   OF  TOXIN   AND   ANTITOXIN 


32 
30 
28 
26 
24 
22 
20 
18 
16 
14 
12 
10 
8 
6 
4 
2 

\ 

\ 

v 

\ 

\ 

(\ 

\ 

"^ 

^ 

^ 

) 

\ 

\ 

\ 

\ 

\ 

f 

\ 

^ 

v 

2    4     6    8    10    12    14    16  -18' 

FIG.  21. — CURVE  OF  NEUTRALIZATION  OF  DIPHTHERIA  TOXIN  (MADSEN). 

This  table  shows  the  difference  between  the  observed  and  calcu- 
lated results : 


Antitoxin. 

Free  Toxin 
(observed). 

Free  Toxin 
(calculated). 

O'l 

75'I 

75*1 

0-I5 

62'6 

627 

0-2 

47-6 

50*6 

0-25 

45-8 

38-6 

0-3 

25*9 

27-3 

o'35 

i7'3 

17*5 

0-4 

9-6 

9'6 

o'45 

5'3 

6-0 

o'5 

3'1 

4'i 

0-6 

1-6 

2-6 

The  conclusion  to  which  Arrhenius  and  Madsen  came  was  that 
toxin  and  antitoxin  react  like  a  weak  acid  and  a  weak  base ;  that 
the  reaction  is  a  reversible  one ;  and  hence  that  the  combination 
of  toxin  and  antitoxin  is  an  unstable  one,  dissociating  into  free 
toxin  and  free  antitoxin. 


86  ARRHENIUS*    AND    MADSEN'S    VIEWS 

On  this  theory  many  facts  which  were  formerly  very  difficult 
to  explain  become  quite  simple.  Take,  for  instance,  the  exact  point 
of  neutralization  of  toxin  by  antitoxin  as  seen  in  the  determina- 
tion of  the  L0  dose  :  this  has  always  been  a  matter  of  great 
difficulty — a  difficulty  formerly  explained  by  assuming  the  last 
substances  to  be  neutralized  are  the  toxons,  which  have  a  very 
feeble  and  indefinite  pathogenic  action.  On  the  physical  chemistry 
theory  the  difficulty  disappears,  because  there  is  no  point  of  exact 
neutralization.  In  spite  of  the  presence  of  an  excess  of  antitoxin, 
some  dissociation  of  the  toxin-antitoxin  molecule  will  always 
occur,  and  the  mixture  will  always  contain  free  toxin,  though  in 
very  small  amount.  Further,  an  exactly  neutralized  mixture  con- 
taining a  few  lethal  doses  of  toxin  is,  of  course,  without  action, 
whereas  a  large  bulk  of  the  same  mixture  may  be  toxic  ;  this 
also  is  readily  explicable.  Then  there  are  some  old  experiments 
of  Buchner's,  which  showed  that  a  mixture  of  tetanus  toxin  and 
antitoxin  which  was  neutral  to  mice  would  produce  tetanus  in 
guinea-pigs,  and  several  others  (Roux's  and  Roux  and  Vaillard's) 
of  similar  nature.  The  explanation  of  these  is  also  easy. 

The  most  interesting  explanation  of  a  previously  known  phe- 
nomenon which  Madsen  offers  in  the  light  of  his  new  theory  is 
that  of  the  immunization  of  animals  by  means  of  a  neutral  mixture 
of  toxin  and  antitoxin.  If  we  follow  Ehrlich,  and  believe  that  the 
compound  is  a  stable  one,  this  is  very  difficult  to  explain.  Madsen's 
solution  is  that  the  mixture  contains  free  toxin,  to  which  the 
immunizing  property  is  due. 

Again,  we  can  also  explain  the  death  of  animals  from  specific 
intoxication  when  it  occurs  in  spite  of  the  presence  of  free  anti- 
toxin in  the  blood  in  exactly  the  same  way.  It  is  true  that  the 
free  antitoxin  would  tend  to  inhibit  dissociation  ;  but,  on  the  other 
hand,  the  hypersensitiveness  of  the  tissues  which  occurs  at  the 
early  stages  of  the  process  of  immunization — and  it  must  be 
remembered  that  it  is  only  in  these  stages  that  death  from  in- 
toxication takes  place — would  render  the  cells  more  susceptible 
to  minute  amounts  of  toxin.  The  explanation  may  not  be  a 
perfect  one,  but  it  appears  to  be  the  best  forthcoming. 

The  importance  of  the  whole  question  from  the  point  of  view 
of  immunity  rests  on  this  question  of  the  dissociability  of  the 
combination,  for  it  is  obvious  that  if  this  is  the  case,  our  views  of 
the  action  of  antitoxin  in  the  animal  body  will  be  very  different 
from  those  we  shall  hold  if  we  regard  the  toxin-antitoxin  molecule 


INTERREACTIONS    OF   TOXIN    AND    ANTITOXIN  87 

as  an  inert  one  of  no  further  interest ;  and  this  theory  of  dissocia- 
tion is  open  to  the  objection  urged  by  Nernst,  and  probably  felt 
by  most  bacteriologists  on  the  first  enunciation  of  the  views  of 
Arrhenius  and  Madsen.     Thus,  if  the  combination  of  toxin  and 
antitoxin  undergoes  dissociation  in  the  living  animal  and  the  toxin 
is  set  free,  it  will  immediately  combine  with  the  susceptible  cells. 
The  equilibrium  will  now  be  disturbed  and  more  toxin-antitoxin 
molecules  will  be  dissociated,  more  toxin  set  free,  and  more  cells 
poisoned ;  and  this  process  will  go  on  until  all  the  toxin  has  been 
passed  on  to  the  cells  and  the  antitoxin  left  free.      Now  this 
dissociation  takes  place  quickly,  so  that  on  this  theory  it  would 
seem  that  the  antitoxin  would  only  interpose  a  very  temporary 
barrier  between  the  cells  and  the  toxin,  the  lethal  action  of  which 
would  be  delayed,  but  in  no  way  inhibited.     We  will  return  to 
this  question  of  dissociation  shortly,  and  meanwhile  state  briefly 
Ehrlich's  objections  to  the  physical-chemical  theory.     In  the  first 
place,  he  points  out  that  if  we  make  a  mixture  of  two  alkaloids, 
of  which  one  is  haemolytic  and  the  other  not,  and  neutralize  them 
by  the  addition  of  a  strong  acid,  the  result  may  be  represented  by 
a  hyperbolic  curve ;  this  is  put  forward  as  a  parallel  experiment 
to  the  neutralization  by  antitoxin  of  a  substance  containing  active 
toxin  and  inert  toxoid.     Secondly,  some  of  the  curves  given  by 
Madsen  and  Arrhenius  do  not  correspond  very  closely  with  the 
observed  results,  and  this  is  especially  the  case  at  their  commence- 
ment and  termination.     At  the  commencement  of  the  curve  it  is 
found  in  many  cases  that  the  addition  of  small  amounts  of  anti- 
toxin does  not  influence  the  toxicity ;  this  is  readily  explained  on 
the  supposition  of  the  existence  of  prototoxoid,  but  hardly  on  any 
other  hypothesis.     The  most  interesting  point,  however,  is  the 
behaviour  of  the  curves  at  the  termination — i.e.,  in  what  Erhlich 
would  call  the  region  of  the  toxons ;  here  Arrhenius  and  Madsen 
usually  found  figures  which  were  lower  than  the  calculated  results. 
Now  Ehrlich  holds  that  traces  of  toxin  do  not  lead  to  paralysis, 
and  if  the  effect  of  a  nearly  neutral  mixture  of  toxin  and  antitoxin 
were  due  to  dissociation  we  should  expect  no  paralysis  to  occur, 
the  toxic  action  being  due  to  toxin,  and  not,  as  Ehrlich  thinks,  to 
toxon.      Madsen  and  Arrhenius  suggest  that  the  action  of  this 
trace  of  antitoxin  may  be  modified  by  the  presence  of  antitoxin  in 
excess.     Further,   Madsen  and  Dreyer   claim  to   have  found   a 
diphtheria  poison,  of  which  small  quantities  would  cause  paralysis 
without  the  addition  of  antitoxin,  so  that  the  question  of  toxon 


88  EVIDENCE    IN    FAVOUR   OF    DISSOCIATION 

would  not  come  in.  And  they  also  showed  that  certain  mixtures 
of  toxin  and  antitoxin  might  act  fatally  on  rabbits  in  a  few  days 
and  cause  only  paralysis  in  guinea-pigs,  but  that  if  a  little  more 
antitoxin  were  added  it  would  act  as  a  toxon  on  rabbits  and  be 
inert  to  guinea-pigs.  To  explain  this,  Ehrlich  had  to  add  yet 
another  body  to  his  list  of  components  of  the  diphtheria  poison, 
and  to  the  proto-,  deutero-,  trito-toxin,  and  toxon  he  added 
toxonoid,  which  is  inert  for  guinea-pigs  and  produces  paralysis 
in  the  rabbit.  The  subject  will  not  be  followed  farther,  but 
enough  has  been  said  to  show  its  extreme  difficulty. 

To  revert  to  the  question  of  dissociation,  Madsen  and  Walbum 
have  brought  forward  some  definite  evidence  of  its  existence,  of 
which  the  more  important  are  the  following.  They  neutralized 
ricin  with  antiricin,  and  to  the  neutral  mixture  added  some  red 
blood-corpuscles.  After  allowing  the  mixture  to  stand  for  some 
time,  the  latter  were  centrifugalized  off,  when  it  was  found  that 
they  had  become  charged  with  ricin  and  the  fluid  containe(i/free 
antiricin.  / 

Secondly,  they  made  use  of  the  fact  that  diphtheria  toxin  is  a 
substance  of  comparatively  small  molecule,  and  can  diffuse 
through  gelatin,  whereas  its  antitoxin  does  so  very  slowly.  They 
prepared  a  neutral  mixture  of  the  two,  and  placed  it  on  the 
surface  of  a  column  of  gelatin,  and  after  some  forty  days'  contact 
examined  slices  at  different  depths,  and  found  free  toxin  at  a 
certain  distance  from  the  surface.  This  they  explained  by  assum- 
ing that  it  had  dissociated  from  the  antitoxin,  and  diffused  down- 
wards into  the  gelatin.  It  was  pointed  out,  however,  that  if  the 
mixture  had  been  allowed  to  stand  for  some  time  the  phenomenon 
did  not  occur ;  it  would  seem,  therefore,  that  the  combination 
takes  place  slowly,  but,  once  formed,  does  not  dissociate. 

There  is,  however,  other  and  independent  evidence  in  favour  of 
the  theory  that  dissociation  of  a  primary  substance  and  its  anti- 
body does  occur.  Thus  Muir  and  Morgenroth  showed  inde- 
pendently that  if  red  corpuscles  are  treated  with  as  much 
amboceptor  as  they  will  take  up,  and  then  mixed  with  normal 
corpuscles,  some  of  the  amboceptor  will  pass  to  the  latter,  and 
on  the  addition  of  a  suitable  complement-containing  serum  the 
whole  may  be  dissolved.  (In  this  experiment  the  red  corpuscles 
correspond  to  the  toxin,  whilst  the  amboceptor  is  the  antibody 
and  corresponds  to  the  antitoxin.) 

Bordet's  explanation  of  the  phenomena  is  quite  different.     Both 


INTERREACTIONS    OF   TOXIN    AND    ANTITOXIN  OQ 

Ehrlich  on  the  one  hand  and  Arrhenius  and  Madsen  on  the  other 
agree  that  the  combination  of  toxin  and  antitoxin  is  a  chemical 
union,  and  takes  place  in  obedience  to  the  laws  of  multiple 
proportions :  a  single  molecule  of  the  one  substance  always  com- 
bines with  the  same  number  of  the  other  on  complete  neutraliza- 
tion. Bordet  denies  this,  and  compares  the  phenomenon  with  the 
absorption  of  a  stain  by  a  colourable  substance.  His  theory  is 
that  a  molecule  of  toxin  requires  many  molecules  of  antitoxin  for 
its  complete  neutralization,  and  that  it  can  be  partially  neutralized 
or  attenuated  by  a  smaller  number.  Thus  he  holds  that  when  a 
small  amount  of  antitoxin  is  added  to  a  large  amount  of  toxin, 
there  is  not  a  mixture  of  free  toxin  and  of  molecules  of  toxin- 
antitoxin  (as  would  occur  if  either  of  the  theories  already  discussed 
was  true),  but  a  uniform  solution  of  toxin  of  diminished  potency. 
Bordet  shows  that  many  of  the  experimental  results  of  other 
observers  are  explicable  on  his  theory,  and  gives  some  new  facts 
in  support  thereof.  For  example,  the  amount  of  an  emulsion  of 
red  blood-corpuscles  which  can  be  haemolyzed  by  the  addition  of 
a  given  quantity  of  haemolytic  serum  can  be  readily  determined. 
We  will  suppose  that  i  c.c.  of  the  serum  just  dissolves  all  the 
corpuscles  in  5  c.c.  of  the  emulsion,  and  no  more.  We  might 
suppose  that  all  the  red  corpuscles  have  all  their  combining 
valencies  exactly  neutralized,  so  that  we  may  regard  them  as  an 
exactly  neutralized  toxin-antitoxin  mixture.  This,  however,  is 
not  the  case,  for  if  we  add  the  haemolytic  serum  in  small  amounts, 
say  0-05  c.c.  at  a  time,  we  shall  find  that  only  a  small  proportion, 
perhaps  2  c.c.,  of  the  same  emulsion  can  be  completely  haemolyzed. 
Thus  each  corpuscle  takes  up  more  haemolysin  in  the  second  case 
than  in  the  first. 

The  objection  may  be  raised  that  a  red  corpuscle  is  very 
different  from  a  molecule  of  toxin.  It  can  undoubtedly  combine 
with  a  vast  number  of  molecules  of  its  antibody  (haemolysin),  but 
it  is  quite  easy  to  understand  how  complete  haemolysis  may  take 
place  if  only  a  certain  number  of  its  combining  valencies  are 
occupied  by  antibody.  But  the  molecule  of  toxin  is,  as  we  have 
already  seen  reason  to  believe,  much  smaller  than  the  molecule 
of  antitoxin ;  and  although  this  fact  does  not  in  itself  render  it 
impossible  that  the  toxin  is  of  higher  valency  than  the  latter, 
it  certainly  renders  it  unlikely  that  it  is  so  much  higher  in  this 
respect  that  there  can  be  an  indefinite  number  of  stages  between 
free  toxin  and  a  fully  neutralized  one.  Bordet  has,  however, 


90  ADSORPTION 

brought  forward  evidence  in  favour  of  his  view  to  which  this 
objection  can  hardly  apply.  It  will  not  be  discussed  here,  as  it 
involves  certain  questions  concerning  serum  haemolysins  which 
have  not  yet  been  discussed. 

Bordet's  theory  supplies  an  explanation,  though  hardly  an 
adequate  one,  of  the  negative  phase.  We  have  already  seen  that 
when  an  animal  is  producing  antitoxin  an  injection  of  toxin  will 
cause  a  fall  in  the  antitoxic  value  of  the  serum  far  greater  than 
can  be  accounted  for  by  the  neutralization  effected  by  the  toxin 
injected  :  it  may  be  2,000  times  as  great,  or  more.  If  we  assume 
that  the  combination  does  not  take  place  in  obedience  to  the  laws 
of  multiple  proportions,  we  can  imagine  that  it  may  go  on  very 
differently  in  the  body,  and  that  in  that  situation  a  small  amount 
of  toxin  may  neutralize  a  large  amount  of  antitoxin.  But  it  is 
very  difficult  to  believe  that  this  is  the  true  explanation,  for  it 
would  involve  an  increase  of  the  neutralizing  power  of  the  toxin 
to  2,000  times  that  which  it  has  outside  the  body — an  increase 
which  certainly  seems  improbable.  Further,  we  have  been  asked 
to  imagine  a  molecule  of  antitoxin  spreading  itself  out  and 
weakening  many  molecules  of  toxin,  and  we  are  now  asked  to 
imagine  a  molecule  of  toxin  dividing  itself  amongst  2,000  molecules 
of  toxin,  and  completely  neutralizing  them. 

The  last  conception  which  we  have  to  consider  is  that  of  Biltz, 
which  approaches  very  closely  to  Bordet's.  Starting  with  the 
idea  that  toxins  and  antitoxins  are  both  colloids,  he  attempts  to 
show  that  their  union  is  analogous  to  adsorption  rather  than  to 
an  ordinary  chemical  union. 

Adsorption  is  a  process  akin  to  solution,  and  does  not  necessarily 
involve  a  chemical  union  between  the  two  substances  which  take 
part  in  it.  It  may  take  place  between  colloids  and  colloids,  or 
between  colloids  and  crystalloids,  and  in  other  ways.  The  two 
substances  taking  part  in  the  process  are  found  to  be  electrically 
positive  and  negative  (as  shown  by  their  moving  to  the  cathode 
or  anode  in  an  electric  field) ;  thus  a  colloidal  solution  of  silicic 
acid  (which  is  negative)  is  precipitated  slowly  by  K2SO4,  which 
has  a  feeble  positive  charge  ;  more  rapidly  by  CuSO4,  which  has  a 
stronger  one  ;  and  immediately  by  A12(SO4)3,  which  has  a  stronger 
one  still. 

Biltz  and  his  collaborators  started  with  the  idea  that  toxins  and 
antitoxins  are  both  colloids.  They  attempted  to  find  the  electrical 
nature  of  tetanus  toxin  by  electrolysis,  but  found  that  it  was 


INTERREACTIONS    OF   TOXIN    AND    ANTITOXIN  QI 

destroyed ;  the  destruction  occurred,  however,  sooner  at  the 
cathode  than  at  the  anode,  suggesting  that  it  was  a  negative 
colloid.  They  found  it  to  be  precipitated  by  positive  colloids, 
such  as  colloidal  hydrated  oxide  of  iron  or  chromium,  though  this 
action  occurred  only  in  vitro,  not  in  vivo.  Tetanus  antitoxin,  how- 
ever, did  not  lose  its  activity  when  exposed  to  mineral  colloids. 

Further,  there  is  an  approach  to  the  phenomenon  of  specificity 
in  the  adsorption  of  one  colloid  by  another,  since  certain  colloids 
are  only  precipitated  by  certain  other  colloids  ;  the  specificity, 
however,  is  by  no  means  so  exact  as  in  the  case  of  the  toxins,  etc., 
and  their  antibodies. 

A  further  analogy  arises  in  the  explanation  of  the  difference 
between  the  L0  and  L+  dose,  which  is  paralleled  by  the  relation- 
ship between  colloidal  ferric  hydroxide  and  arsenious  acid.  When 
these  two  substances  are  mixed  adsorption  and  precipitation  take 
place ;  hence  the  use  of  the  former  substance  as  an  antidote  for 
the  latter.  Now  it  is  found  that  if  a  solution  of  arsenious  acid  is 
just  rendered  tox*e  by  the  addition  of  ferric  hydroxide,  very  much 
more  than  one  lethal  dose  of  arsenic  must  be  added  to  make  the 
mixture  toxic  again. 

The  further  investigation  into  the  evidence  which  they  have 
adduced  would  lead  us  too  far  into  the  study  of  the  agglutinins  to 
be  undertaken  here.  It  will  be  dealt  with  subsequently  (see 
Chapter  XII.). 

It  seems,  on  the  whole,  that  no  theory  is  absolutely  sufficient  to 
explain  all  the  phenomena,  and  that  as  soon  after  each  new  one  is 
adduced  the  supporters  of  the  older  ones  bring  forward  evidence 
which  renders  it  untenable.  The  probability  is,  at  the  time  of 
writing,  that  Ehrlich's  views  are  generally  held,  and  are  open  to 
the  fewest  objections.  They  are  complicated,  it  is  true,  and  have 
had  to  undergo  constant  modifications  as  new  facts  have  arisen  ; 
but  the  facts  themselves  are  complicated.  Yet  it  must  be  con- 
fessed that  there  are  some  grave  objections  to  its  acceptance  in  its 
present  form,  and  it  may  become  yet  more  involved  before  it  can 
be  fully  accepted  as  a  complete  explanation.  Thus  the  analogy 
with  other  bodies,  and  the  phenomena  of  the  death  from  intoxica- 
tion of  animals  with  antitoxin  in  their  blood,  seem  to  point  strongly 
to  the  theory  that  the  toxin-antitoxin  molecule  dissociates  strongly 
both  in  vivo  and  in  vitro.  Yet  this  is  not  compatible  with  Ehrlich's 
views. 


CHAPTER  V 

THE  ORIGIN  OF  ANTITOXIN— THE  SIDE-CHAIN 

THEORY 

THE  theory  that  antitoxins  are  derived  from  their  appropriate 
toxins  deserves  a  short  consideration,  since  it  is  so  inherently 
probable.  When  we  consider  the  large  numbers  of  toxins  which 
give  rise  to  their  antitoxins  when  injected  into  animals,  and  see 
that  each  antitoxin  has  a  direct  action  on  its  own  toxin,  but  none 
or  practically  none  on  others,  the  most  likely  explanation  of  the 
phenomenon  is  that  the  animal  tissues  have  the  power  of  splitting 
the  toxin  into  two  parts,  or  of  otherwise  modifying  it,  and  that 
this  modified  toxin  can  combine  with  unaltered  toxin  and  form 
antitoxin.  Some  experimental  evidence  is  forthcoming  to  support 
this  view.  Thus  it  is  found  that  diphtheria  toxin  which  has  been 
submitted  to  the  action  of  an  electric  current  loses  its  toxicity,  but 
retains  that  of  producing  immunity  on  injection.  Some  thought 
that  this  was  due  to  the  direct  transformation  of  the  toxin  into 
"  artificial  antitoxin,"  and  it  was  hoped  that  a  similar  change 
might  be  brought  about  in  the  human  body  in  disease  by  the  use 
of  electricity.  The  explanation  of  .the  phenomenon  is  very 
simple.  The  electrolysis  of  the  water  in  which  the  toxin  is 
dissolved  gives  rise  to  oxidizing  substances  which  transform  the 
toxins  into  toxoids,  the  immunizing  virtues  of  which  we  have 
already  seen.  There  is  no  method  by  which  antitoxin  can  be 
prepared  other  than  by  the  injection  of  toxins  into  suitable 
animals. 

That  antitoxin  is  not  a  direct  transformation  product  of  toxin 
appears  probable  from  the  following  considerations,  none  of  which 
perhaps  is  conclusive,  but  which  together  make  up  a  body  'of 
evidence  of  some  importance  : 

i.  The  injection  of  a  certain  amount  of  toxin  will  give  rise, 
under  suitable  circumstances,  to  the  formation  of  much  more 

92 


THE    ORIGIN    OF   ANTITOXIN — THE    SIDE-CHAIN    THEORY      93 

antitoxin  than  it  can  neutralize.  Thus  Knorr  showed  that  a 
single  unit  of  toxin  might  call  forth  100,000  units  of  antitoxin. 
This  can  scarcely  be  accounted  for  by  supposing  the  transforma- 
tion of  the  one  substance  into  the  other. 

2.  It  frequently  happens  that   antitoxin  occurs,  sometimes  in 
considerable   amounts,    in   the   blood   of   animals  that  have  not 
received   injections   of   toxin,  and   have   not  suffered,  as  far  as 
known,  from  the  disease  in  question — in  other  words,  of  "  normal  " 
animals.     Thus  horses  frequently  have  traces  of  diphtheria  anti- 
toxin  in   the   blood,  as  much  as  4  units  per  c.c.  having   been 
observed.     Metchnikoff  suggests   that   this   may  be  due   to   the 
action    of   "  pseudo-diphtheria "    bacilli,   which   are  so  common. 
But  no  one  has  been  able  as  yet  to  produce  diphtheria  toxin  or 
symptoms  of  diphtheria  by  means  of  these  bacilli,  and  it  is  not 
usual   to   regard  them  as  having  any  connection,  other  than  a 
superficial  morphological  resemblance,  with  the  true  diphtheria 
bacillus. 

If  we  may  bring  other  antibodies  into  the  argument  the  case 
against  Metchnikoff's  supposition  is  enormously  strengthened. 
The  blood  of  many  animals  contains  agglutinins  and  hsemolysins — 
often  in  large  amounts,  often  under  conditions  in  which  we  may 
almost  exclude  the  possibility  of  a  preceding  infection.  Thus  the 
blood  of  a  normal  horse  will  almost  invariably  clump  typhoid 
bacilli,  B.  pyocyaneus,  the  cholera  vibrio,  and  many  other 
organisms. 

3.  An   animal   which   has   received   an  injection  of  antitoxin 
rapidly  eliminates  it  from  the  blood,  whereas  the  antitoxin  which 
is  formed  in  the  body  remains  a  much  longer  time.     This  argu- 
ment does  not  carry  very  much  weight,  since,  as    Metchnikoff 
points  out,  the   toxin  might  remain   for   long   periods   occluded 
in   the   cells,  and   only  undergo   a  gradual   transformation   into 
antitoxin. 

4.  Roux  and  Vaillard  showed  that  the  whole  of  an  animal's 
blood  might  be  removed  by  repeated  venesections,  and  that  the 
newly  regenerated  blood  might  contain  almost  as  much  antitoxin 
as  was  present  before  the  haemorrhage,  thus  suggesting  a  new 
formation.     But  the  objection  urged  under  the  last  heading  is  of 
weight  here  also,  and  if  we  consider  it  probable  that  toxin  may 
remain   latent   in  the  tissues  for   long   periods  without   causing 
symptoms,   we   shall  exclude   this   piece   of   evidence   from  the 
argument,  as  well  as  the  observations  of  Salomonsen  and  Madsen, 


94  CONSTITUTION    OF    PROTOPLASM 

that  the  production  of  antitoxin  may  be  increased  by  an  injection 
of  pilocarpin. 

If  we  reject  this  explanation  of  the  phenomenon,  and  regard 
the  production  of  antitoxin  as  being  analogous  to  the  process  of 
internal  secretion,  we  must  regard  it  as  being  due  to  the  action  of 
the  living  cells  when  they  are  stimulated  by  the  toxin.  There  is 
at  the  present  time  but  one  theory  which  suggests  how  this  may 
be  brought  about :  this  is  Ehrlich's  celebrated  side-chain  theory, 
which  has  played  so  important  a  part  in  the  modern  study  of 
problems  concerning  infection  and  immunity,  and  which,  though 
highly  hypothetical,  deserves  a  close  and  careful  study. 

Ehrlich  points  out  that  a  cell  has  two  functions.  The  first 
is  the  physiological  function,  which  it  enacts  in  the  body — secretory 
in  the  case  of  a  gland  cell,  conductive  in  the  case  of  a  nerve  fibre, 
etc. ;  and  the  second  is  the  function  of  nutrition,  necessary  to 
supply  the  waste  which  is  constantly  taking  place.  We  must 
suppose  a  similar  constitution  for  each  of  the  complex  molecules 
of  living  protoplasm  which  build  up  the  cell ;  in  each  molecule 
there  is  a  portion  which  discharges  the  specific  function  of  the 
cell,  and  this  portion  has  to  be  nourished.  The  functional  portion 
is  the  more  important,  and  is  called  by  Ehrlich  the  active  centre 
(Leistungskern).  In  our  diagrams  we  shall  represent  it  as  forming 
the  central  portion  of  the  giant  molecule,  but  it  is  hardly  necessary 
to  caution  the  reader  that  this  diagrammatic  representation  has 
no  necessary  morphological  basis.  It  is  just  as  permissible  to 
regard  this  portion  as  diffused  through  the  nutritive  part  of  the 
molecule  as  long  as  the  difference  in  constitution  and  action  of  the 
two  are  borne  in  mind. 

The  second  portion,  that  concerned  in  nutrition,  is  more 
important  in  relation  to  immunity.  We  may  regard  it  as  having 
two  functions — it  "  seizes  "  suitable  molecules  of  food  substances 
from  the  blood  or  lymph  in  which  it  is  bathed,  and  it  alters  them 
in  such  a  way  as  to  build  them  up  into  living  material  which  can 
take  its  place  in  the  molecule  of  protoplasm,  so  as  to  replace  that 
which  has  been  used  in  the  life  of  the  cell. 

The  function  of  "  seizing  "  molecules  of  food  from  the  sur- 
rounding tissues  implies  a  selective  faculty,  for  we  cannot  imagine 
that  all  the  molecules  which  circulate  in  the  blood  and  lymph  are 
suitable  for  all  cells  at  all  times.  This  apparently  intelligent 
selection  of  suitable  material  is,  of  course,  at  bottom  a  chemical 
process  :  the  food  molecule  becomes  attached  to  some  portion  of 


THE    ORIGIN    OF   ANTITOXIN— THE    SIDE-CHAIN    THEORY      95 

the  cell  for  which  it  has  a  chemical  affinity.  Now  Ehrlich 
supposes  that  this  affinity  for  food  molecules  is  situated  in  certain 
portions  of  the  molecule  of  protoplasm — in  certain  groups  of  atoms 
which  he  calls  side-chains,  receptors,  or  haptines.  [The  name  "  side- 
chain  "  was,  perhaps,  badly  chosen.  It  denotes  a  possible  analogy 
with  complex  organic  bodies — e.g.,  of  the  aromatic  group — which 
are  composed  of  a  central  portion  (such  as  the  benzene  ring)  and 
side-chains,  on  which  many  of  their  reactions  depend.  The  com- 
parison is  not  a  very  close  one,  and  all  that  is  necessary  is  to 
regard  the  molecule  of  protoplasm  as  possessing  numerous  groups 
of  atoms,  each  of  which  has  an  affinity  for  one  of  the  bodies 
circulating  in  the  body  fluids,  and  necessary  for  the  life  of  the 
molecule  in  question.] 

On  this  theory  the  nutrition  of  the  molecule  takes  place  as 
follows  :  A  molecule  of  suitable  food  substance  in  the  fluid  sur- 
rounding the  cell  is  brought  into  contact  with  one  of  the  receptors 
for  which  it  has  a  chemical  affinity,  and  the  two  unite.  This 
is  the  first  step  in  the  process.  The  food  is  "anchored  "  to  the 
cell  by  means  of  a  receptor,  for  which  it  has  a  specific  combining 
affinity,  or  which,  to  use  Ehrlich's  analogy,  fits  it  like  a  key  fits  a 
lock.  The  second  stage  involves  a  process  which  we  may  compare 
to  digestion,  by  which  the  food  molecule  is  altered  in  some  pro- 
found manner,  and  absorbed,  in  whole  or  in  part,  into  the  molecule 
of  protoplasm. 

Let  us  apply  this  theory  to  the  process  by  which  a  cell  is 
poisoned.  We  will  imagine  for  a  moment  that  the  molecule  of 
toxin  contains  a  group  of  atoms  which  will  unite  specifically  with 
the  side-chain  of  one  of  the  body  cells.  We  have  already  shown 
that  this  molecule  of  toxin  contains  a  haptophore  group  of  mole- 
cules, in  virtue  of  which  it  can  combine  with  antitoxin,  and  a 
toxophore  group,  on  which  its  toxic  action  depends.  We  can  now 
go  farther,  and  say  that  the  first  stage  of  the  intoxication  of  a  cell 
by  means  of  a  true  toxin  consists  in  the  union  of  the  haptophore 
group  of  atoms  in  the  toxin  to  a  receptor  of  a  molecule  of  proto- 
plasm, this  receptor  being  one  which  "  fits  it  like  a  key  fits  a 
lock."  Each  molecule  of  protoplasm  has  innumerable  receptors, 
of  which  only  a  certain  number  are  suitable  for  this  toxin.  This 
is  the  first  step.  The  toxin  molecule  is  now  "  anchored  "  to  the 
living  cell,  and  in  the  second  stage  the  toxophore  radicle  of  the 
toxin  comes  into  play.  We  may  regard  this  toxophore  group  as 
exerting  an  enzyme-like  action  on  the  protoplasm  through  the 


96  INTOXICATION    AND    NUTRITION 

haptophore  group  and  the  receptor,  by  which  the  two  are  united. 
Thus: 


CELL 


FIG.  22. — CELL  MOLECULE  WITH  RECEPTORS  (E,  E). 

A,  A,  Molecules  of  toxin  (B  =  toxophore,  C  =  haptophore),  D  =  a  molecule 

of  toxoid. 

The  result  of  this  is  that  the  protoplasm  is  poisoned.  If  only  a 
few  of  its  receptors  are  united  to  toxin  molecules  the  result  may 
be  but  slight,  whereas  if  more  are  occupied  the  functioning 
centre  of  the  molecule  will  be  affected,  and  marked  symptoms  will 
arise,  and  if  still  more  molecules  of  toxin  are  linked  up  the  mole- 
cule of  protoplasm  may  be  killed. 

The  essential  point  in  this  process  is  that  it  is  exactly  analogous 
to  natural  nutrition,  except  that  in  the  latter  case  the  receptor 
unites  with  a  molecule  which  is  of  use  to  the  cell,  whereas  in  the 
former  it  unites  with  one  which  simply  resembles  the  food  molecule 
in  having  a  haptophore  group  with  similar  chemical  affinities. 
Thus,  intoxication  with  bacterial  toxins  is  essentially  a  process  of 
nutrition,  but  is  perverted  in  its  later  stages  by  the  nature  of  the 
toxophore  group  of  the  toxin. 

Now  consider  the  case  in  which  a  molecule  of  protoplasm  is 
attacked  by  a  number  of  molecules  of  toxin  which  is  not  large 
enough  to  kill.  A  considerable  number  of  receptors  will  be  taken 
up  by  toxin,  and  we  must  consider  these  receptors  as  being  thereby 
rendered  useless.  The  protoplasm,  however,  has  need  of  these 
receptors,  and  the  need  may  probably  be  more  pressing  since  it  is 
poisoned,  and  metabolic  changes  may  take  place  more  rapidly,  and 
there  is  thus  greater  need  for  renewed  nutriment.  Fresh  receptors 
must  therefore  be  formed,  and  Ehrlich  compares  their  regeneration 
to  the  budding  forth  of  fresh  tentacles  in  the  hydra,  to  replace 


THE    ORIGIN    OF  ANTITOXIN — THE    SIDE-CHAIN   THEORY      97 

those  which  have  been  lost.  Now  suppose  a  fresh  dose  of  toxin 
reaches  the  cell,  and  that  these  new  receptors  are  in  their  turn 
taken  up  by  toxin,  but  yet  not  in  numbers  sufficient  to  kill  the 
cell.  The  same  processes  will  occur :  the  receptors  will  be  rendered 
useless,  and  a  fresh  crop  of  the  same  nature  will  be  produced. 

Now  in  accordance  with  the  well-known  laws  of  habit,  in  virtue 
of  which  a  part  of  the  body  can  gradually  be  trained  to  perform  a 
function  which  is  difficult  at  first,  but  which  becomes  more  easy 
by  usage,  the  molecule  of  protoplasm  gradually  acquires  the 
faculty  of  producing  these  receptors  more  and  more  easily,  in 
obedience  to  nicely  graduated  doses  of  toxin.  If  these  are 
repeated  in  proper  amount  and  at  suitable  intervals  the  cell  may 
be  trained,  so  to  speak,  to  produce  these  receptors,  and  it  may 


FIG.  23. 

ultimately  come  to  do  so  in  excess  and  far  beyond  its  physiologi- 
cal requirements.  This  also  is  analogous  with  known  biological 
facts,  such  as  the  production  of  callus  in  a  fractured  bone  in 
excess  of  the  amount  necessary  for  repair.  We  shall  revert  to 
this  point  later.  Now  we  have  to  imagine  that  these  receptors 
may  be  formed  in  excess  so  great  that  they  cannot  all  remain 
attached  to  the  cell ;  some  of  them  are  pushed  off,  so  to  speak,  by 
the  younger  ones,  which  are  growing  up  to  take  their  place.  If 
this  occurs,  these  receptors  will  pass  off  freely  into  the  blood.  As 
we  have  seen,  they  are  so  constituted  that  they  will  unite  speci- 
fically with  the  particular  toxin  that  was  injected.  These  cast-off 
receptors  constitute  antitoxin,  and  the  formation  of  that  substance  is 
simply  due  to  the  pathological  separation  of  the  groups  of  atoms, 
in  virtue  of  which  the  toxin  molecule  is  linked  up  to  the  living 

7 


98 


WEIGERT'S  HYPOTHESIS 


protoplasm.  The  whole  process  is  explained  as  one  of  perverted 
nutrition,  and  (once  admitting  Ehrlich's  theory  of  the  constitution 
of  the  molecule  of  protoplasm  and  of  the  way  it  is  nourished) 
without  introducing  any  but  well-recognized  biological  pheno- 
mena. The  theory  is  fascinating  in  its  simplicity,  and  although 
there  are  a  few  difficulties  in  the  way  of  its  full  acceptance  as  a 
complete  explanation  of  the  facts,  it  certainly  accounts  for  them 
far  better,  on  the  whole,  than  any  other  theory  will  do  —  if,  indeed, 
an  alternative  one  has  been  put  forth.  And  it  has  the  great  merit 
in  a  theory  that  its  application  has  led  to  the  discovery  of  new 
facts.  * 

A  few  remarks  are  necessary  in  connection  with  the  question 


FIG.  24. 

of  the  mechanism  in  which  the  over-production  of  side-chains  is 
produced.  Weigert's  hypothesis  with  regard  to  the  nature  of 
hyperplasia  as  the  result  of  injury  or  irritation  is  now  well  known, 
and  a  brief  outline  will  suffice.  He  imagines  that  the  maintenance 
of  the  normal  structure  and  physiological  function  of  a  tissue 
depends  upon  a  condition  of  equilibrium  brought  about  by  a 
series  of  mutual  restraints  exerted  by  each  cell  on  its  neighbours. 
In  the  same  way,  the  structure  of  a  single  cell,  or  perhaps  of  a 
single  compound  molecule  of  protoplasm,  depends  on  a  similar 
equilibrium  existing  between  minute  living  units.  If  one  of  these 
components  is  killed  or  injured,  the  restraint  which  it  exerts  on 
the  surrounding  units  is  removed,  and  unrestrained  growth  can 


THE    ORIGIN    OF   ANTITOXIN — THE    SIDE-CHAIN    THEORY      QQ 

take  place ;  and  Weigert  points  out  that  this  always  goes  on  to 
excess,  more  new  material  being  formed  than  is  necessary  to 
replace  the  amount  lost.  The  application  of  this  theory  to  the 
mode  of  new  formation  of  receptors  is  sufficiently  obvious. 

We  will  now  consider  certain  phenomena  in  the  light  of  the 
side-chain  theory. 

The  occurrence  of  antibodies  in  the  blood  of  normal  animals  is 
susceptible  of  a  ready  explanation.  The  receptors  are,  in  general, 
united  to  the  cell,  but  it  is  easily  conceivable  that  a  few  may  be 
desquamated  accidentally  and  escape  into  the  blood.  It  must  be 
remembered  that  the  actual  amount  (weight  or  bulk)  of  these 
antibodies  in  the  normal  blood  is  probably  always  infinitesimal, 
although  the  effects  of  this  small  amount  may  be  striking.  Thus 
the  agglutination  of  typhoid  bacilli  at  a  dilution  of  i  to  160 
(which  often  occurs  with  the  blood  of  normal  horses)  indicates 
the  presence  of  a  very  small  amount  of  specific  antibody,  as  is 
apparent  from  the  fact  that  the  blood  may  be  made  to  clump  at 
i  to  1,000,000  without  being  altered  chemically  in  any  way  that 
can  be  detected.  If  an  exceedingly  minute  proportion  of  all  the 
receptors  are  shed  into  the  blood,  it  will  probably  account  for  all 
the  antibodies  found  in  that  situation  under  normal  conditions. 

The  fact  that  toxoids  produce  antitoxin1  is  also  explicable.  We 
must  regard  the  receptor  which  is  occupied  with  toxoid  as  being 
thereby  rendered  useless  for  the  protoplasm,  although  the  latter  is 
not  poisoned  as  a  result  of  the  union. 

Next  arises  a  most  important  question,  and  one  which  is  ap- 
parently satisfactorily  answered  on  the  side-chain  theory — the 
fact  that  certain  substances  (bacterial  toxins  in  particular)  give 
rise  to  the  production  of  antibodies,  whilst  others,  such  as  the 
alkaloids,  glucosides,  mineral  poisons,  poisons  of  simple  con- 
stitution, such  as  alcohol,  etc.,  do  not.  (This  may  be  taken  as 
fairly  proved,  certain  researches  which  go  to  prove  the  existence 
of  antibodies  to  morphine,  alcohol,  etc.,  being  inconclusive.) 

The  explanation  is  founded  on  differences  in  the  way  the 
various  substances  form  combinations  with  the  protoplasm.  This 
latter  has,  as  an  integral  part  of  its  constitution,  certain  receptors 
which  have  a  specific  combined  affinity  for  proteids,  and  we  must 
imagine  the  union  of  proteids,  whatever  be  their  nature,  as  taking 
place  by  a  direct  union  with  these  receptors.  Other  substances, 

1  According  to  Ehrlich.  The  fact  is  not  universally  admitted,  though  all 
agree  that  they  will  produce  immunity. 

7—2 


IOO  NATURE    OF    UNION    OF   TOXINS   AND    TISSUES 

however,  need  not,  and  in  all  probability  do  not,  unite  with  the 
protoplasm  in  this  way.  Ehrlich,  basing  his  theories  on  the  fact  that 
certain  alkaloids  and  other  substances  can  be  extracted  from  their 
combinations  with  organic  matter  by  simple  means,  has  suggested 
that  the  union  of  non-proteid  poisons  with  protoplasm  is  less  firm 
than  in  the  other  case.  This  seems  doubtful,  for  nothing  could 
be  much  firmer  than  the  combination  of  nitrate  of  silver  or  similar 
bodies  with  the  tissues.  And  we  know  as  a  matter  of  fact  that 
the  toxin-protoplasm  compound  is  not  necessarily  a  stable  one. 
Emulsions  of  tissues  will  abstract  tetanus  toxin,  and  retain  it  on 
washing,  but  give  it  up  again  in  a  free  state  when  allowed  to  stand 
for  some  time  in  contact  with  normal  saline  solution.  However 
this  may  be — and  the  point  is  not  of  importance — we  may  readily 
admit  with  Ehrlich  that  it  is  highly  probable  that  proteids  unite 
with  protoplasm  in  a  manner  fundamentally  different  from  alka- 
loids, etc.  The  former  process  follows  on  physiological  lines, 
whilst  the  latter  is  a  pathological  process  entirely,  and  one  which 
has  no  counterpart  in  normal  nutrition.  The  former  may  be 
compared  to  the  insertion  of  the  key  in  the  lock,  the  latter  to  the 
violent  smashing  of  the  lock.  If  this  is  the  case,  it  is  easy  to 
see  that  "chemical"  poisons  cannot  be  expected  to  give  rise  to 
the  production  of  antibodies.  It  is  true  that  if  a  cell  is  already 
secreting  antibodies,  we  might  expect  that  a  substance  which 
stimulates  the  general  metabolism  of  the  cell  and  increases  the 
rapidity  of  the  vital  processes  might  temporarily  increase  this 
production,  and  we  have  seen  that  this  is  the  case  with  pilo- 
carpin,  which  stimulates  the  production  of  diphtheria  antitoxin  in 
an  immunized  horse.  This  is  an  entirely  different  process,  and 
one  that  is  easily  explicable  on  the  side-chain  theory. 

It  is  necessary,  therefore,  to  determine  whether  all  the  sub- 
stances which  give  rise  to  the  production  of  antibodies  on  injec- 
tion are  proteids.  We  must  admit  at  the  outset  that  in  many  cases 
this  cannot  be  proved ;  the  chemical  constitution  of  toxins,  enzymes, 
and  many  other  antigens  is  as  yet  quite  unknown.  But  in  the 
cases  in  which  the  chemical  composition  of  the  primary  substance 
is  ascertained  we  find  it  to  be  invariably  of  proteid  nature ;  thus, 
solutions  of  any  of  the  coagulable  proteids  will  give  rise  to  the 
production  of  precipitins;  the  proteid  substances  present  in  the 
body  of  bacteria  give  rise  to  the  production  of  agglutinins,  which 
are  themselves  of  a  proteid  nature,  and  will,  on  injection  into 
suitable  animals,  give  rise  to  anti-agglutinins  ;  the  proteid  con- 


THE    ORIGIN    OF   ANTITOXIN — THE    SIDE-CHAIN    THEORY      IOI 

stituents  of  the  red  blood-corpuscles,  the  substances  forming  their 
stroma,  call  forth  the  production  of  haemolysins  ;  and  a  great  many 
other  cases  might  be  quoted.  It  may  be  taken  as  definitely 
established  that  wherever  the  nature  of  the  antigen  is  known  and 
falls  into  one  of  the  known  groups,  that  antigen  is  a  proteid. 

We  may  almost  go  a  step  farther,  and  say  that  all  proteids,  of 
whatever  nature,  when  injected  into  suitable  animals,  will  give 
rise  to  antibodies  ;  in  fact,  some  writers  actually  enunciate  this 
as  a  law.  There  are,  however,  a  few  exceptions,  which  are 
gradually  diminishing  in  face  of  further  researches,  and  it  is 
highly  probable  that  time  will  show  that  the  law  is  valid  to  the 
fullest  extent. 

Reverting  now  to  the  question  of  the  nature  of  the  bodies  of 
unknown  constitution  which  form  antibodies — i.e.,  the  toxins, 
enzymes,  etc. — we  may  ask  whether  these  are  not  in  reality 
proteids,  in  spite  of  their  failure  to  give  the  proteid  reactions  as 
usually  accepted  by  chemists.  The  question  is  largely  one  of 
nomenclature.  Accepting  the  definitions  usually  given  in  physio- 
logical textbooks,  and  based  on  a  study  of  ordinary  proteids,  such 
as  egg-albumin,  etc.,  we  may  admit  that  they  are  not;  but  are 
these  definitions  valid  ?  Might  we  not  define  them  with  greater 
accuracy  as  substances  from  which  animal  organisms  can  obtain 
food  material  containing  combined  ("  organic  ")  nitrogen  ?  In 
this  case  we  should  substitute  for  coarse  chemical  experiments 
in  vitro  the  more  delicate  reactions  of  the  living  animal  body; 
any  nitrogenous  substance  which  is  recognized  by  living  proto- 
plasm as  being  a  suitable  pabulum  for  it  would  be  defined  as  a 
proteid.  If  this  is  the  case,  we  are  thrown  back  at  once  on 
Ehrlich's  theory,  and  can  define  the  proteids  as  substances  which 
unite  in  a  specific  manner,  haptophore  to  receptor,  with  the  living 
molecule  of  protoplasm.  If  we  do  this,  we  may  admit  that  the 
toxins  and  enzymes,  whatever  their  chemical  reactions,  and 
whether  they  are  or  are  not  food  substances  for  the  cells,  combine 
with  protoplasm  in  the  manner  which  is  characteristic  of  proteids, 
and  are  to  be  regarded  as  proteids  when  looked  at  from  this  point 
of  view.  And  we  must  not  forget  that  many  of  the  substances 
which  we  regard  as  toxins  are,  as  a  matter  of  fact,  of  value  in 
nutrition  to  the  animal  which  forms  them,  and  only  act  as  poisons 
in  strange  species.  Thus,  the  ichthyotoxin  of  eel  serum  must 
nourish  the  cells  of  the  eel,  but  is  a  powerful  toxin  to  almost 
all  other  species.  Whether  any  animal  can  utilize  tetanus  toxin, 


IO2  CONDITIONS    OF   ANTIBODY    PRODUCTION 

diphtheria  toxin,  etc.,  as  cell  pabulum  is  doubtful,  but  there  are 
some  experiments  which  go  to  show  that  this  is  the  case.  Thus, 
the  rat  is  relatively  immune  to  diphtheria  toxin,  and  shows  no 
symptoms  after  the  injection  of  an  amount  which  is  lethal  for 
many  rabbits,  and  this  immunity  is  not  due  to  the  presence  of 
antitoxin.  This  being  so,  it  seems  clear  that  the  diphtheria  toxin 
disappears  from  the  blood  of  the  rat  in  virtue  of  forming  a  com- 
bination with  the  cells  of  the  latter  without  injuring  them,  and  we 
may  fairly  assume  that  it  is  of  value  in  nutrition,  although,  of 
course,  this  assumption  is  not  open  to  direct  proof.  In  a  similar 
way  tetanus  toxin  disappears  rapidly  from  the  blood  of  scorpions, 
no  antibody  being  formed. 

Thus  it  appears  to  be  a  logical  sequel  of  the  acceptance  of 
Ehrlich's  theory  that  we  may  define  proteids  as  substances  which, 
when  injected  into  suitable  animals,  give  rise  to  the  production  of 
antibodies.  We  have  now  to  glance  for  a  moment  at  the  ques- 
tion of  the  suitability  of  different  animals  for  the  production  of 
antibodies. 

Researches  with  precipitins  show  that  the  proteid  substances 
which  are  present  in  the  serum  of  any  species  of  animal  are 
different  from  those  which  occur  in  any  other.  We  must  regard 
the  molecule  of  proteid  of  any  type  (say  of  a  globulin)  as  being  of 
great  complexity,  and  capable  of  many  slight  modifications,  which 
are  inappreciable  to  gross  chemical  tests,  but  which  are  perfectly 
obvious  to  the  living  animal  cells.  Human  globulin  differs  from 
horse  globulin,  and  this,  again,  from  sheep  globulin.  Further, 
some  facts  go  to  show  that  differences  exist  between  the  proteids 
of  animals  of  the  same  species,  and  that  the  proteids  in  the  serum 
of  one  man  or  horse  are  not  exactly  the  same  as  those  in  another 
man  or  horse.  These  differences  come  in  as  a  result  of  the  last 
step  in  the  process  of  digestion  ;  the  food  materials  are  broken 
down  into  simpler  bodies  in  the  alimentary  canal,  and  built  up 
again  in  their  passage  through  the  epithelial  cells  which  line  that 
structure,  and  in  this  process  receive  certain  distinctive  features 
peculiar  to  the  species  or  to  the  animal  in  question.  As  a  result, 
the  body  fluids  of  any  animal  contain  proteids  which  have  been 
deliberately  adapted  for  the  nutrition  of  the  cells  of  that  animal, 
but  which  may  be  quite  useless  or  even  toxic  for  those  of  another 
species.  The  prime  requisite  of  their  suitability  for  nutritive 
purposes  is  that  the  proteid  molecules  should  possess  haptophores 
which  "  fit "  the  receptors  of  the  cell  molecules ;  the  second, 


THE    ORIGIN   OF   ANTITOXIN — THE   SIDE-CHAIN    THEORY      103 

equally  important,  is  that  the  proteid  molecules  shall  be  "  diges- 
tible "  by  the  cell.  If  the  first  requisite  fails,  the  proteid  molecule 
fails  to  unite  with  the  cells  ;  it  may  remain  for  long  periods  in  the 
blood  (as  is  the  case  with  tetanus  toxin  in  the  blood  of  Oryctes, 
where  it  remains  months  after  injection),  or  may  probably  be 
eliminated  with  the  excretions,  or  be  otherwise  dealt  with.  In 
this  case  no  antibody  can  be  formed. 

When  the  injected  proteid  molecule  finds  suitable  receptors  and 
unites  with  them,  but  when  the  resulting  compound  is  not  suit- 
able for  the  nutrition  of  the  cell,  and  cannot  be  "  digested  "  by  it, 
the  case  is  different.  The  receptors  which  are  occupied  by  the 
molecules  of  proteid  are  useless  for  the  cell,  and  the  conditions 
which  we  have  discussed  in  dealing  with  the  action  of  toxins  are 
reproduced,  although  there  may  be  no  toxic  action  ;  the  receptors 
are  useless  and  have  to  be  regenerated,  and  under  suitable  circum- 
stances may  be  produced  in  excess  and  form  antibodies.  The 
criteria  of  the  suitability  of  any  animal  for  the  production  of  an 
antibody  to  a  given  proteid  are  therefore  three  in  number  : 

1.  This  proteid  must  not  exist  naturally  in  the  blood  of  the 
animal. 

2.  It  must  possess  haptophores  which  "  fit "  the  receptors  of 
the  animal  cells. 

3.  The  compound  thus  formed  must  be  "  indigestible  "  by  the 
cell  and  useless  for  its  nutrition. 

These  are  the  theoretical  criteria,  which  are  derived  from  a 
study  of  the  side-chain  theory,  and,  on  the  whole,  experimental 
research  agrees  with  them,  and  thus  corroborates  the  theory  to  an 
extraordinary  extent.  For  example,  we  need  scarcely  refer  to 
criterion  i  :  we  know  that  precipitins  do  not  occur  normally  in 
the  blood  of  any  animal  to  the  proteids  which  circulate  in  that 
fluid ;  it  is  only  the  injection  of  a  foreign  proteid  which  causes 
their  production.  Further,  the  more  closely  allied  are  two  animal 
species,  the  less  the  production  of  precipitin  when  serum  from  the 
one  is  injected  into  the  other.  Thus,  in  order  to  prepare  a  potent 
antihuman  serum,  the  blood  of  a  man  must  be  injected  into  a 
rabbit,  fowl,  etc.,  not  into  a  monkey.  If  it  is  true,  as  appears  to 
be  the  case,  that  the  serum  of  an  animal  causes  the  production  of 
precipitins  when  injected  into  another  of  the  same  species,  this 
does  not  affect  the  argument,  for  these  precipitins  only  occur  in 
minute  amount,  and  simply  show  that  the  fluids  and  tissues  of 
different  animals  of  the  same  species  are  not  in  reality  identical. 


IO4  CONDITIONS    OF   ANTIBODY    PRODUCTION 

There  is  nothing  surprising  in  that  when  we  consider  how  different 
persons  vary  in  constitution  and  susceptibility.  Again,  it  is  not 
uncommon  to  find  auto-agglutinins  in  human  blood — i.e.,  sub- 
stances which  agglutinate  human  red  corpuscles.  When  this  is 
the  case,  it  is  found  that  these  agglutinins  have  no  action  on  the 
red  corpuscles  of  the  persons  from  whom  the  serum  is  taken,  but 
only  on  those  of  other  individuals ;  the  stroma  of  the  corpuscles 
cannot  act  as  an  antigen  to  the  cells  with  which  it  comes  normally 
into  contact.  (The  way  in  which  these  auto-agglutinins  are 
called  into  existence,  and  their  meaning,  if  any,  or  use  in  the 
economy,  are  still  unexplained.) 

The  existence  and  mode  of  formation  of  the  secondary  anti- 
bodies— i.e.,  the  antibodies  to  antibodies — point  in  the  same 
direction.  Thus,  if  we  inject  typhoid  bacilli  or  their  proteid 
constituents  into  rabbits  or  other  animals,  the  specific  antibody — 
the  agglutinin — is  produced,  and  accumulates  in  the  blood  in  con- 
siderable amount,  but  it  does  not  produce  anti -agglutinin.  If, 
however,  we  inject  this  serum  into  an  animal  of  another  species, 
and  preferably  one  far  removed  from  the  first  zoologically,  the 
production  of  anti -agglutinin  occurs.  The  agglutinin  produced  in 
any  one  species  of  animal  must  have,  in  addition  to  its  peculiar 
clumping  properties,  the  general  constitution  of  a  proteid  charac- 
teristic of  that  animal,  and  hence  be  devoid  of  the  power  of 
forming  antibodies  whilst  in  the  blood  where  it  was  produced. 
Similar  phenomena  occur  in  the  production  of  other  haemolysins 
(amboceptor),  which  will  be  referred  to  subsequently. 

With  regard  to  the  second  criterion — the  possession  of  hapto- 
phores  which  fit  the  receptors  of  the  protoplasm  of  the  animal 
into  which  it  is  injected — it  is  only  necessary  to  say  that,  as  far 
as  can  be  traced,  the  substances  which  produce  antibodies  do,  as  a 
matter  of  fact,  disappear  from  the  blood  of  the  animal  in  a  short 
time— a  few  hours  or  days.  As  has  been  pointed  out,  the  converse 
of  this  is  not  necessarily  true ;  the  toxin  may  disappear  from  the 
blood,  and  yet  no  antitoxin  be  formed,  as  in  the  case  of  tetanus 
toxin  in  scorpions.  Here  we  have  assumed  that  the  scorpion 
contains  naturally  a  proteid  with  a  haptophore  closely  allied  to 
that  of  tetanus  toxin,  that  the  toxophore  of  the  latter  is  without 
action  on  the  cell  with  which  it  is  linked,  and  that  the  latter  can 
use  the  former  as  pabulum  in  the  same  way  as  it  uses  the 
molecules  of  normal  occurrence  in  its  blood. 

The  third  criterion  is  that  the  proteid  molecule  shall  not  be 


THE   ORIGIN    OF   ANTITOXIN-— THE    SIDE-CHAIN   THEORY      105 

capable  of  assimilation  by  the  protoplasm  to  which  it  is  anchored ; 
it  is  not  necessary  that  there  should  be  a  toxophore  group  which 
injures  the  cell.  In  this  case  the  production  of  the  antibody  will 
not  be  accompanied  by  any  symptoms  of  disease.  The  proto- 
plasmic molecule  will  have  some  of  its  receptors  occupied  and 
rendered  useless  by  the  foreign  proteid,  and  will  have  to  regenerate 
others  of  the  same  nature,  but  it  will  not  be  injured  in  any  way  in 
the  process.  It  is  in  this  way  that  we  explain  the  formation  of 
antitoxins  as  a  sequence  to  the  injection  of  toxoids  which  have 
retained  their  haptophore  groups,  but  lost  their  toxophores,  as 
well  as  the  formation  of  agglutinins  (to  non-pathogenic  bacteria), 
precipitins,  etc. 

We  pass  on  to  another  question — that  of  specificity.  We  have 
seen  that  the  antitoxins  are,  in  general,  adapted  only  to  neutralize 
the  toxins  which  lead  to  their  production.  To  this  rule  there  are 
a  few  exceptions,  real  or  apparent.  Thus,  antirobin  serum  has 
also  an  action  against  ricin,  tetanus  antitoxin  has  apparently 
some  action  on  snake-venom,  anti-snake  venom  serum  neutralizes 
scorpion  venom,  etc.  The  explanation  is  doubtless  that  the 
venoms  in  question  have  haptophore  groups  which  closely  re- 
semble one  another  in  their  chemical  affinities,  whilst  differing 
from  those  of  other  toxins.  Thus,  Ehrlich  has  suggested  that 
robin  is  the  toxon  of  ricin,  in  which  case  the  combining  portions 
of  the  molecules  of  the  two  would  be  identical. 

In  any  case,  the  side-chain  theory  seems  to  fit  quite  well  with 
ascertained  facts.  It  explains  the  specificity  ;  it  is  the  receptors 
which  unite  with  the  injected  toxin  which  are  produced  in  excess, 
and  which  therefore  form  antitoxin.  But  in  the  complex  changes 
in  metabolism  which  must  take  place  in  the  poisoned  cell  it  is 
quite  easy  to  imagine  that,  under  certain  circumstances,  other 
receptors  may  either  be  formed,  in  slight  excess  as  a  result  of  the 
general  stimulation  of  the  cell,  or  may  be  cast  off  in  slight  excess 
as  a  result  of  necrotic  or  autolytic  processes  taking  place  therein. 
This  may  be  the  explanation  of  some  of  the  apparent  exceptions, 
especially  of  those  in  which  the  serum  has  but  slight  antitoxic 
power  on  the  toxin  which  was  not  injected,  whereas  it  is  much 
more  potent  on  that  which  was. 

The  next  question,  and  a  much  more  difficult  and  important 
one,  deals  with  the  site  of  production  of  the  antibodies.  Ehrlich's 
original  idea  was  that  the  antitoxins  are  produced  from  those  cells 
on  which  the  toxins  act — i.e.,  on  the  susceptible  cells.  For  instance, 


I06  TETANUS   TOXIN    AND    BRAIN-TISSUE 

in  the  case  of  tetanus,  he  supposed  that  antitoxin  is  formed  by  the 
cells  in  the  central  nervous  system,  and  explained  the  great  difficulty 
of  immunizing  animals  to  this  toxin  by  pointing  out  the  enormous 
susceptibility  of  the  cells  to  the  action  of  this  poison ;  it  is  only 
in  the  cells  of  the  central  nervous  system  that  antitoxin  can  be 
formed,  and  these  cells  are  extremely  easily  killed  by  the  toxin, 
and  are  necessary  for  life.  The  chief  evidence  in  favour  of  this 
theory  is  derived  from  the  experiments  of  Wassermann  and  of 
Romer,  which  we  shall  now  consider  seriatim. 

Wassermann's  experiment  may  be  regarded  as  a  corollary  to 
that  of  Ransom,  who  found  that,  after  injection  of  tetanus  toxin 
in  pigeons  (which  are,  in  the  ordinary  sense  of  the  word,  in- 
susceptible to  that  substance),  he  could  extract  it  from  all 
substances  except  the  brain.  Wassermann  and  Takaki  found 
that  the  mixture  of  tetanus  toxin  with  an  emulsion  of  the  central 
nervous  system  was  no  longer  toxic  when  injected  into  susceptible 
animals ;  in  other  words,  that  the  emulsion  of  central  nervous 
system  behaved  like  antitoxin.  Emulsions  of  other  organs  are 
devoid  of  this  power.  They  will  combine  with  tetanus  toxin,  but 
will  yield  it  again  when  injected  into  susceptible  animals  or 
macerated  in  salt  solution.  It  appears,  therefore,  that  the  central 
rjervous  system  is  the  seat,  and  the  only  seat,  of  an  antitoxin-like 
substance,  and  it  is  thus  rendered  at  least  probable  that  when 
antitoxin  appears  in  the  blood  it  is  due  to  its  release  from  the 
cells  which  contain  it  normally — in  other  words,  from  the  cells  of 
the  central  nervous  system,  the  cells  whichX  attacks.  The  case 
of  tetanus  is  the  only  one  in  which  a  toxin  appears  to  have  a 
definite  selective  influence  on  one  group  of  cells,  and  is  therefore 
unique  in  providing  a  means  whereby  Ehrlich's  supposition  as 
to  the  origin  of  antitoxin  may  be  tested.  It  is  no  wonder  that 
Wassermann's  experiment  has  been  submitted  to  an  extra- 
ordinarily careful  investigation,  the  results  of  which  we  must 
outline  briefly.  Some  of  these  experiments  seem  to  point  to  the 
truth  of  Ehrlich's  assumption.  Thus,  Blumenthal  showed  that 
after  injection  of  tetanus  toxin  into  living  animals  the  spinal  cord 
lost  its  property  of  fixing  the  toxin  in  vitro,  the  receptors  of  the 
cells  being  already  occupied  with  that  substance.  Further,  boiled 
brain  substance  loses  its  power  of  neutralizing  toxin,  just  as  anti- 
toxin does  on  being  heated.  He  also  showed  that  the  central 
nervous  system  of  fowls,  which  are  but  slightly  susceptible  to 
tetanus,  has  but  little  power  of  binding  that  toxin,  so  that  emul- 


THE    ORIGIN    OF    ANTITOXIN —THE    SIDE-CHAIN    THEORY      107 

sions  of  the  brain  must  remain  in  contact  with  the  toxin  for  some 
time  before  the  latter  is  neutralized. 

It  was  urged  by  many  writers  that  the  brain  cannot  be  the  site 
of  production  of  antitoxin,  since  the  injection  of  tetanus  toxin 
direct  into  the  brain  of  immunized  animals  causes  the  develop- 
ment of  tetanic  symptoms.  This  argument  is  obviously  fallacious. 
A  cell  that  is  secreting  antitoxin  still  possesses  side-chains  suitable 
for  union  with  the  toxin ;  it  possesses  them,  indeed,  in  increased 
amount,  and  is  therefore,  if  anything,  more  susceptible  to  the 
action  of  the  toxin.  The  antitoxin  which  is  circulating  in  the 
blood  only  prevents  the  toxin  from  reaching  the  cell,  acting  much 
in  the  same  way  as  a  lightning  conductor. 

Further  research,  however,  seems  to  prove  definitely  that  the 
neutralizing  properties  of  brain  substance  are  not  due  to  the 
presence  of  a  substance  having  any  real  likeness  to  antitoxin. 
Thus,  Metchnikoff  showed  that  when  an  emulsion  of  frog's  brain 
is  mixed  with  tetanus  toxin  no  neutralization  takes  place,  although 
the  frog  is,  under  certain  circumstances,  susceptible  to  the  action 
of  the  toxin  ;  these  researches  were  corroborated  by  Courmont 
and  Doyon.  Arguing  from  this  and  other  facts,  Metchnikoff 
attributed  the  fixation  fo  tetanus  -as*itoxin  by  the  central  nervous 
system  to  the  presence  in  the  latter  of  fatty  substances  ;  these  are 
absent  from  the  brain  of  the  frog.  In  support  of  this  view,  several 
observers  found  that  substances  such  as  lecithin,  cholesterin, 
tyrosin,  etc.,  have  also  the  power  of  neutralizing  various  toxins. 

It  was  objected  to  these  researches  of  Metchnikoff  and  Cour- 
mont and  Doyon  that  these  experimenters  did  not  leave  the 
tetanus  toxin  in  contact  with  the  brain  substance  for  a  sufficiently 
long  time.  But  this  seems  unnecessary,  since  the  emulsion  of  the 
central  nervous  system  of  higher  animals  will  neutralize  tetanus 
toxin  if  the  mixture  be  injected  immediately,  or  even  if  the 
two  substances  are  injected  separately,  though  in  this  case  the 
necessary  amount  of  brain  substance  is  larger.  And  Danysz 
showed  that  if  a  neutral  mixture  of  brain  and  toxin  be  macerated 
for  a  long  time  in  salt  solution  the  toxin  is  again  set  free,  in  which 
again  the  phenomenon  is  unlike  that  presented  by  antitoxin  in  its 
action  on  toxin. 

The  researches  of  Morax  and  Marie  also  point  in  the  same 
direction.  They  showed  that  the  fixative  power  of  the  brain  is 
almost  entirely  destroyed  by  drying,  whereas,  as  we  know,  that 
process  has  practically  no  effect  on  antitoxin.  The  researches  of 


io8  ROMER'S  EXPERIMENTS 

Dmitrevsky  are  also  of  great  interest,  and  almost  constitute  a 
crucial  experiment. 

If  Ehrlich's  theory  is  true,  and  if  antitoxin  is  produced  by  the 
budding-off  of  the  receptors  in  great  numbers,  it  ought  to  follow 
that  the  brain  of  an  immunized  animal,  which  contains  these 
receptors  in  abnormally  great  amount,  ought  to  have  a  greater  power 
of  neutralizing  toxin  than  that  of  normal  animals.  But  Dmitrev- 
sky's  experiments  show  that  this  is  not  the  case,  and  constitute  a 
strong  proof  against  the  theory  in  its  original  form.  It  seems  fair 
to  conclude  that  Wassermann's  phenomenon  is  due  to  some 
accidental  property  of  the  central  nervous  system,  or  possibly  to 
the  presence  of  fats,  and  must  not  be  quoted  as  evidence  of  the 
origin  of  antitoxin  from  the  cells  on  which  it  chiefly  acts. 

Romer's  experiment  was  made  with  abrin,  a  substance  which 
has  the  power  of  causing  violent  conjunctivitis.  He  made  use 
of  rabbits,  instilling  small  but  increasing  amounts  into  the  right 
conjunctiva.  After  three  weeks  the  animal  was  killed,  and  each 
conjunctiva  dissected  off,  ground  up  with  one  lethal  dose  of  abrin, 
and  injected  into  animals.  The  right  conjunctiva,  that  into  which 
the  abrin  had  been  instilled,  was  found  to  act  as  an  antitoxin  and 
to  neutralize  the  abrin,  whereas  the  left  was  devoid  of  this  power. 
Of  course,  if  the  process  were  carried  on  for  a  long  time  the  abrin 
would  be  absorbed  into  the  system,  and  there  would  be  a  general 
production  of  antitoxin,  but  at  first  the  process  appears  local. 

These  researches  are  interesting,  but  do  not  seem  to  have  any  very 
direct  bearing  on  the  question  at  issue.  They  show — what  does 
not  require  proof — that  tissues  which  are  not  reached  by  the  toxin 
do  not  produce  antitoxin  ;  but  the  question  as  to  whether  the 
latter  substance  is  produced  by  the  cells  which  are  peculiarly 
susceptible  to  the  toxin  is  not  elucidated.  There  are  in  the 
conjunctiva  numerous  structures — connective-tissue  cells,  blood- 
vessels, endothelium,  epithelium,  leucocytes,  etc.  —  and  these 
experiments  afford  no  means  of  gauging  which  of  these  are 
affected  by  the  toxin  or  which  produce  antitoxin.  The  behaviour 
of  the  leucocytes  is  of  especial  interest ;  they  are  present  in  the 
inflamed  conjunctiva  in  increased  amounts,  and  the  question  may 
be  asked  whether  the  apparent  antitoxic  action  of  the  right  con- 
junctiva may  not  be  due  to  these  cells,  which  are  present  in  but 
small  numbers  in  the  left.  This  view  is  rendered  more  probable 
from  the  further  researches  of  Romer,  who  showed  that  in 
immunized  animals  antiabrin  is  present  in  greater  amounts  in  the 


THE    ORIGIN    OF   ANTITOXIN — THE    SIDE-CHAIN    THEORY 

organs  rich  in  leucocytes,  such  as  the  spleen,  lymph  glands,  and 
bone-marrow.  This  view  harmonizes  well  with  that  of  Metch- 
nikoff,  which  we  shall  consider  subsequently. 

The  chief  arguments  against  the  origin  of  antibodies  from  the 
cells  which  are  especially  acted  on  are  those  of  Metchnikoff  on 
the  production  of  antispermotoxin,  and  from  the  study  of  other 
cytotoxins.  The  first  series  of  experiments  are  of  fundamental 
importance,  and  will  be  discussed  here,  although  the  substances  in 
question  are  not  simple  bodies  like  toxins,  but  have  a  much  more 
complex  structure.  The  main  facts  are  as  follows  :  The  injection 
of  spermatozoa  into  living  animals  is  followed  by  the  production 
and  appearance  in  the  serum  of  certain  substances  which  have 
the  power  of  immobilizing  and  clumping  the  spermatozoa  of  the 
species  from  which  the  spermatozoa  used  for  the  injection  was 
taken  ;  we  may  call  the  substances  spermotoxin.  If  the  serum  of 
animals  which  has  been  prepared  in  this  way  is  now  injected  into 
animals  of  another  species,  the  result  will  be  the  formation  of  an 
antispermotoxin  analogous  with  antitoxin,  which  inhibits  the 
action  of  the  spermotoxic  serum  in  vitro  on  the  spermatozoa. 

Now  on  Ehrlich's  theory  we  may  assume  that  this  anti- 
spermotoxin is  derived  from  the  cells  from  which  the  spermotoxin 
is  derived — i.e.,  from  the  spermatozoa.  But  Metchnikoff  proved 
that  this  is  not  the  case,  since  the  substance  is  developed  as  the 
result  of  the  injection  of  spermotoxic  serum  into  castrated  males 
or  into  young  animals  of  either  sex.  Antispermotoxins,  therefore, 
are  not  necessarily  derived  from  sperm  cells.  This  is  certainly  a 
very  striking  result,  and  one  that  tells  against  the  side-chain 
theory,  but  it  is  not  conclusive  for  this  reason — the  cytotoxins  are 
not  usually  sharply  specific.  Thus  a  cytotoxic  serum  made  by  the 
injection  of  kidney  cells  has  a  profound  action  on  the  kidney,  but 
it  usually  has  some  haemolytic  action  also,  and  may  affect  other 
cells  as  well.  There  do  not  seem  to  be  any  experiments  bearing 
on  the  point,  but  it  is  at  least  probable  that  spermotoxins  have 
some  action  on  cells  other  than  spermatozoa,  and  if  this  is  the 
case  Metchnikoff 's  experiments,  considered  as  a  proof  against  the 
side-chain  theory,  fall  to  the  ground. 

The  mode  of  formation  of  the  antihaemolysins  also  calls  for  short 
notice  in  this  connection.  The  haemolysins  are,  in  some  cases  at 
least,  more  sharply  specific  than  some  of  the  cytotoxins,  acting 
apparently  almost  exclusively  on  the  red  blood-corpuscles.  It 
should  follow  on  Ehrlich's  theory  that  the  antihaemolysins  are 


IIO  SITE   OF  ANTITOXIN    FORMATION 

derived  mainly  from  the  red  blood-corpuscles.  This  view, 
however,  seems  very  difficult  to  believe  when  we  consider  that 
these  substances  are  devoid  of  protoplasm  and  nuclei,  and  do  not 
present  any  of  those  appearances  indicative  of  active  metabolism 
or  secretion  which  we  should  expect  in  the  case  of  a  cell  perform- 
ing so  profound  a  physiological  function  as  the  production  of  an 
antitoxin. 

Nor  is  there  any  evidence,  except  in  the  one  doubtful  case  of 
the  action  of  brain  substance  on  tetanus  aa^koxin,  of  the  presence 
of  substances  like  antibodies  in  the  normal  tissues  and  organs,  in 
spite  of  the  fact  that  they  must  contain  receptors  which  would 
neutralize  the  toxins  to  which  they  are  tested.  Thus  Calmette 
did  not  find  that  emulsions  of  brain  possessed  any  neutralizing 
action  on  snake  venom,  although  that  substance  certainly  acts  on 
the  central  nervous  system,  and  there  are  many  similar  researches 
on  the  actions  of  other  emulsions  of  organs  on  other  toxins,  all 
with  negative  results.  We  may  quote,  for  example,  those  of  Blum, 
who  submitted  various  organs  of  the  horse — liver,  spleen,  etc.,  all 
of  which  are  certainly  acted  on  by  diphtheria  toxin,  since  they 
show  definite  morphological  lesions  in  acute  cases  of  fatal  intoxi- 
cation— to  prolonged  autolysis,  and  found  nothing  in  the  nature  of 
an  antitoxin  for  diphtheria  or  snake  venom  in  the  resulting 
material.  In  one  case — that  of  autolyzed  lymphatic  glands  of  the 
calf — he  found  a  substance  which  reacted  like  tetanus  antitoxin. 

On  the  whole,  therefore,  it  would  seem  that  there  is  no  good 
evidence  for  the  theory  that  the  cells  which  are  acutely  and 
powerfully  poisoned  by  means  of  toxin  are  those  from  which  the 
antitoxin  is  derived.  It  is  certain,  however,  that  this  substance 
must  be  formed  from  cells  which  are  reached  and  affected  in  some 
way  by  the  toxin.  The  cells  which  especially  require  discussion 
are  those  of  the  connective  tissues  and  the  leucocytes. 

Attention  is  drawn  especially  to  the  connective  tissues  from  the 
fact  that  the  most  powerful  production  of  antitoxin  is  usually 
elicited  by  a  subcutaneous  injection  of  toxin ;  intraperitoneal 
injection  is  in  general  less  potent,  and  intravenous  injection  less 
still,  and  may  not  be  followed  by  any  production  of  antitoxin 
at  all.  The  same  law  holds,  and  even  more  strongly,  in  the 
production  of  the  other  antibodies,  although  some  exceptions 
occur. 

Now  in  some  cases  it  is  true  that  these  toxins  have  a  local 
pathogenic  action  on  the  tissues  into  which  they  are  injected,  but 


THE    ORIGIN    OF   ANTITOXIN — THE    SIDE-CHAIN    THEORY      III 

in  others  this  is  extremely  slight,  or  not  more  than  that  due  to 
the  injection  of  any  so-called  "  inert  "  fluid  into  that  situation. 
Consider  in  this  connection  the  reactions  of  certain  animals  to 
tetanus  toxins.  When  this  substance  is  injected  into  guinea-pigs, 
the  effect  is  practically  the  same  whether  the  injection  is  sub- 
cutaneous or  intracerebral.  In  rabbits,  on  the  other  hand, 
a  very  much  smaller  dose  suffices  to  kill  if  injected  into  the  brain 
than  is  necessary  if  the  injection  is  subcutaneous.  Now  rabbits 
are  less  susceptible  to  tetanus  toxin  than  are  guinea-pigs,  requiring 
relatively  larger  doses  to  bring  about  a  fatal  issue.  On  the  other 
hand,  it  is  easier  to  prepare  tetanus  antitoxin  from  rabbits  than 
from  guinea-pigs  by  the  injection  of  tetanus  toxin,  though  difficult 
in  both  cases.  Compare  these  results  with  those  obtained  from 
fowls.  These  animals  are  resistant  against  tetanus  toxin  (except 
in  absolutely  enormous  doses)  when  injected  subcutaneously,  but 
are  easily  affected  when  the  injection  is  made  into  the  brain 
or  subarachnoid  space.  The  probable  explanation  turns  on  the 
varying  affinity  of  the  toxin  for  different  parts  of  the  body.  We 
may  assume  in  all  cases  that  the  tetanus  toxin  acts  as  a  poison 
only  on  the  brain  and  spinal  cord,  and  that  in  the  guinea-pigs  it 
has  practically  no  affinity  for  the  subcutaneous  tissues,  and 
that  when  injected  into  this  situation  it  passes  ultimately  to  the 
brain  almost  without  loss.  In  rabbits  some  of  the  toxin  is  united 
to  the  tissues,  and  only  a  part  reaches  the  central  nervous  system, 
whilst  in  fowls  the  affinity  of  the  tissues  for  the  toxin  is  so  great 
that  the  whole  of  the  latter  is  absorbed  and  the  brain  escapes 
injury.  Now  it  is  practically  impossible  to  obtain  antitoxin  by 
injecting  toxin  into  the  guinea-pig,  difficult  to  obtain  it  from  the 
rabbit,  but  easy  to  obtain  it  from  the  fowl,  so  that  it  would  appear 
that  it  is  the  tissues  which  are  not  especially  vulnerable  to  the 
toxin  which  yield  it.  If  this  is  true,  it  is  no  argument  against  the 
validity  of  the  side-chain  theory.  It  simply  indicates  that  the 
cells  which  are  profoundly  affected  with  toxin  are  thereby 
rendered  unable  to  produce  antitoxin  in  virtue  of  the  toxic  action 
of  the  poison,  the  other  conditions  being  suitable.  We  know  this 
to  be  the  case  from  the  fact  that  we  can  produce  tetanus 
antitoxin  from  guinea-pigs  as  a  result  of  the  injection  of  toxoid, 
which  we  must  assume  to  unite  entirely  with  the  cells  of  the 
central  nervous  system,  just  as  the  toxin  does,  though  without 
injuring  them.  We  may  assume,  therefore,  that  antitoxin  is 
produced  from  cells  which  unite  with  toxin  or  toxoid,  but  which 


112  SIGNIFICANCE    OF    LEUCOCYTOSIS 

are  not  too  profoundly  injured  thereby  to  perform  their  functions 
of  self -nutrition  in  a  normal  manner. 

The  question  whether  we  can  narrow  down  the  issue  to  one 
particular  set  of  cells  which  always  occur  in  the  connective  and 
other  tissues — to  wit,  the  leucocytes  —is  a  more  difficult  one.  We 
have  already  adverted  to  the  researches  of  Romer,  who  found  that 
antiabrin  was  present  in  immunized  animals  in  greatest  amount 
in  the  organs  that  are  rich  in  leucocytes,  and  Metchnikoff  has 
brought  forward  a  whole  series  of  researches  which  point  strongly 
in  the  same  direction.  The  discussion  of  some  of  these  will  be 
deferred  to  a  subsequent  chapter,  since  they  hardly  seem  to 
indicate  that  the  leucocytes  form  antitoxin,  though  they  do  go 
to  prove  that  they  discharge  another  and  equally  important 
function  in  the  production  of  immunity  to  toxins.  Here  it  will 
only  be  necessary  to  point  out  some  well-known  and  important 
facts  which  appear  to  have  a  bearing  on  the  question  of  the 
origin  of  antitoxin.  We  allude  especially  to  the  chemotactic 
attraction  for  leucocytes  which  is  showrn  by  almost  all  toxins 
when  not  present  in  too  great  an  amount.  In  virtue  of  this 
attraction  the  most  marked  and  constant  feature  of  intoxication 
is  the  presence  of  an  excess  of  leucocytes,  and  this  is,  in  general, 
most  marked  when  there  is  a  balanced  contest  between  the  toxin 
and  the  host — i.e.,  when  the  latter  is  neither  destroyed  at  once 
nor  shows  but  little  effect  of  the  injection.  It  is  practically  a 
general  rule  that  wherever  the  host  is  victorious  in  the  struggle 
with  the  toxin  there  is  an  excess  of  leucocytes ;  and  even  where 
the  fatal  issue  is  not  averted  there  is  some  leucocytosis,  unless 
the  intoxication  is  a  very  grave  one  and  very  rapidly  fatal.  This 
is  well  known  in  human  pathology  in  the  infective  processes, 
where  a  diminution  of  the  number  of  leucocytes  is  a  bad  sign, 
whereas  hyperleucocytosis  is  a  good  sign  in  so  far  as  it  indicates 
that  the  infection,  though  intense,  is  being  combated  by  the 
patient  by  the  best  means  at  his  disposal.  Many  researches  have 
indicated  that  the  same  law  holds  in  these  intoxications.  Thus 
Chatenay,  in  an  exhaustive  study  on  the  reactions  of  the  more 
important  toxins,  finds  that  a  dose  that  is  rapidly  fatal  brings 
about  a  fall  in  the  leucocytes,  whereas  smaller  doses  cause  hyper- 
leucocytosis;  thus,  in  the  guinea-pig  about  100  lethal  doses  are 
required  to  prevent  the  occurrence  of  the  latter.  Now  we 
can  scarcely  imagine  that  so  widespread  and  constant  a  phe- 
nomenon is  without  some  meaning  or  without  some  use  to  the 


THE    ORIGIN    OF   ANTITOXIN — THE    SIDE-CHAIN    THEORY       113 

organism,  and  since  it  always  occurs  and  is  most  marked  under 
conditions  in  which  antitoxin  is  formed,  it  is  highly  probable  that 
the  leucocytes  may  be,  entirely  or  in  part,  the  origin  of  the 
antitoxin.  But  we  must  not  forget  that  in  many  cases  the 
leucocytes  play  other  parts  in  the  struggle  against  the  infections ; 
for  example,  it  is  plausible  to  argue  that  their  presence  in  excess  in 
infected  areas  is  an  attempt  to  combat  the  bacteria  themselves,  and 
not  the  toxins  which  they  produce.  In  this  case  their  access  to 
areas  into  which  a  toxin  had  been  injected  might  be  useless,  and  they 
might  simply  be  attracted  there  under  the  mistaken  supposition  that 
there  would  be  bacteria  to  combat  as  well  as  toxins.  But  this 
view  is  rendered  improbable  in  face  of  the  numerous  facts  that 
have  been  brought  out  by  the  French  school  with  regard  to  the 
absorption  of  toxins  and  other  poisons  by  the  leucocytes.  Thus 
Metchnikoff  showed  (and  it  is  a  fact  of  the  utmost  importance) 
that  if  an  aseptic  exudate  be  produced  in  a  fowl  that  has  been 
injected  with  tetanus  toxin,  that  .substance  can  be  demonstrated 
in  large  amount  in  the  leucocytes  of  the  exudate.  Again,  Vaillard 
and  Vincent  have  shown  that  if  tetanus  bacilli  or  spores  washed 
free  from  toxin  are  injected  into  a  guinea-pig,  they  become 
surrounded  by  leucocytes,  and  that  under  such  circumstances  no 
tetanic  symptoms  occur. 

With  regard  to  the  action  of  other  poisons  the  facts  are  still 
*•  more  striking.  Thus  rabbits  are  far  more  susceptible  to  the 
v  intracerebral  injection  of  atropin  than  to  the  same  substance 
injected  into  the  circulation;  and  when  the  injection  has  been 
made  in  the  second  manner,  atropin  can  be  demonstrated  from 
the  leucocytes,  whereas  it  is  not  present,  or  only  in  very  small 
amounts,  in  the  plasma  and  red  corpuscles.  Similar  facts  have 
been  found  for  arsenic  and  other  poisons.  Still  more  striking  are 
the  researches  of  Besredka  with  regard  to  arsenic.  He  studied 
first  the  action  of  the  trisulphide  of  arsenic,  an  almost  insoluble 
substance,  yet  very  poisonous.  When  this  was  injected  in  small 
amounts  into  the  peritoneal  cavity  of  a  guinea-pig,  there  was  a 
marked  increase  in  the  mononuclear  leucocytes  in  that  situation, 
and  in  a  short  time  these  cells  had  ingested  the  whole  of  the 
arsenic,  which  could  be  recognized  as  fine  granules  in  their 
protoplasm.  These  gradually  became  smaller  and  ultimately 
disappeared,  being  probably  converted  into  a  non-toxic  compound 
— perhaps  by  some  process  analogous  with  antitoxin  formation. 
In  a  second  series  of  experiments  he  tested  the  action  of  the 

8 


114         ACTION  OF  LEUCOCYTES  ON  TOXINS 

same  substance  when  shielded  from  the  leucocytes  by  being 
enclosed  in  permeable  bags  before  being  placed  in  the  peritoneum ; 
in  this  case  the  animal  died,  the  arsenic  doubtless  becoming 
dissolved,  and  thus  escaping  the  action  of  the  leucocytes.  He 
found  also  that  substances  which  diminished  the  number  of  the 
leucocytes  in  the  peritoneal  fluid  aided  the  action  of  the  arsenic, 
whilst  those  which  caused  a  leucocytosis  diminished  it.  He  found 
similar  facts  with  relation  to  the  action  of  a  soluble  salt  of  arsenic, 
and  showed  that  in  animals  which  had  been  immunized  to  that 
substance  the  arsenic  is  especially  taken  up  by  the  leucocytes. 
There  can  be  no  doubt,  therefore,  that  the  leucocytes  play  a  role 
of  the  utmost  importance  in  the  defence  of  the  body  against  the 
ordinary  poisons. 

All  things  being  considered,  we  may  deduce  that  antitoxin  is 
formed  from  any  cell  with  which  the  toxin  can  combine,  provided 
that  that  cell  is  not  too  profoundly  injured  in  the  process,  and 
may  attribute  to  the  leucocytes  an  important,  though  not  an 
exclusive,  role  in  this  process.  It  is  hardly  necessary  to  say  that 
this  does  not  disprove  the  side-chain  theory ;  we  must  regard  the 
leucocytes  as  being  cells  which  are  specially  told  off  for  the 
defence  of  the  organism  against  infections  and  intoxications. 
They  are,  in  consequence,  specially  immune  to  the  action  of 
toxins,  being  able  to  resist  amounts  which  are  injurious  to  the 
more  highly  organized  tissues ;  that  this  is  the  case  is  proved 
by  their  invasion  and  persistence  in  a  living  state  in  areas  in 
which  the  tissues  are  profoundly  injured  by  a  toxin.  It  is  not 
difficult  to  believe  that  these  resistant  cells,  which  have  a  special 
predilection  for  intoxicated  areas  in  which  antitoxin  is  especially 
required,  should  be  the  source  of  that  substance.  We  have,  then, 
only  to  regard  them  as  possessing  suitable  haptophore  groups  and 
as  being  relatively  insusceptible  to  the  toxophore  groups — both  of 
which  are  inherently  probable — to  see  that  they  fulfil  in  a  special 
way  the  criteria  which  the  side-chain  theory  demands  for  cells 
which  are  to  produce  antitoxin  when  exposed  to  the  action  of 
toxins. 


CHAPTER  VI 
IMMUNITY  TO  TOXINS 

THE  mechanisms  by  which  the  injurious  effects  of  toxin  in  the 
blood  and  tissues  are  combated  are  exceedingly  complex,  and 
may  be  divided  roughly  into  two  series.  In  the  first  and  less 
interesting,  though  perhaps  not  less  important,  the  mechanism 
may  be  called  (for  want  of  a  better  term)  non-specific.  It  acts 
equally  on  all  or  many  toxins,  and  recovery  from  the  effects  of  the 
poison  is  not  necessarily  or  usually  followed  by  any  immunity 
thereto.  For  example,  the  dilatation  of  the  vessels  and  accelera- 
tion of  the  blood-stream  which  takes  place  in  an  inflamed  focus 
may  be  regarded  as  one  of  the  natural  provisions  for  avoiding  the 
too  intense  action  of  a  toxin  in  one  small  area.  The  poison  pro- 
duced is  rapidly  swept  into,  and  diluted  by,  the  whole  blood- 
stream, and  if  not  produced  in  large  amounts  may  not  reach  any 
tissue  in  amount  sufficient  to  cause  much  damage.  Perhaps,  also, 
the  proteolytic  action  of  the  enzymes  frequently  produced  (mainly 
from  the  polynuclear  leucocytes)  in  the  inflammatory  focus  are  of 
some  importance.  Toxins  are  very  fragile  bodies,  and  it  is  ex- 
tremely probable  that  the  potent  enzymes  which  occur  in  all  fluids 
rich  in  polynuclear  leucocytes  may  bring  about  their  destruction  in 
considerable  amounts. 

The  more  purely  chemical  processes  are  hardly  known  in  their 
application  to  true  toxins,  but  they  have  been  largely  studied  in 
regard  to  the  methods  by  which  the  organism  combats  deleterious 
substances  produced  in  the  course  of  metabolism,  or  absorbed 
from  the  alimentary  canal.  The  latter  are  for  the  most  part 
bacterial  products,  though  not  true  toxins.  It  is,  however,  quite 
possible  that  these  latter  substances  are  destroyed  by  the  same 
processes,  which  Herter  classifies  as  (i)  oxidation,  (2)  hydration 
and  dehydration,  and  (3)  various  syntheses. 

As  an  example  of  the  process  of  oxidation,  we  may  consider  the 
formation  of  indol,  a  toxic  substance,  which  is  formed  by  B.  coli 

"5  8—2 


Il6  NEUTRALIZATION    OF   POISONS 

and  other  organisms  in  the  alimentary  canal,  and  which  probably 
plays  some  part  in  the  production  of  the  symptoms  of  auto-intoxi- 
cation due  to  abnormal  digestive  processes  occurring  in  the  small 
intestine.  It  undergoes  oxidation  to  indoxyl,  a  much  less  toxic 
substance,  which  then  combines  with  sulphate  of  potash  to  form 
indoxyl-potassium-sulphate,  or  indican.  There  is  reason  to  believe 
that  this  process  takes  place  to  a  very  large  extent  in  the  liver, 
one  of  the  chief  functions  of  which  is  the  destruction  or  neutraliza- 
tion of  poisonous  substances  conveyed  to  it  by  the  portal  circula- 
tion. Thus  the  toxic  dose  of  nicotin  and  of  some  other  alkaloids 
is  much  less  when  the  injection  is  made  into  the  general  circulation 
than  if  the  substance  be  injected  into  the  portal  vein.  This,  it 
must  be  noticed,  is  not  due  merely  to  the  elimination  of  the  poison 
in  the  bile,  though  that  may  happen.  The  liver  cells  have  the 
power  of  actually  destroying  certain  of  the  alkaloids.  Thus 
hyoscyamine  mixed  with  surviving  liver  pulp  is  rapidly  destroyed. 
In  the  same  way,  indol,  phenol,  and  skatol,  all  toxic  substances  of 
bacterial  origin,  cannot  be  recovered  by  distillation  after  contact 
with  liver  cells.  It  appears,  therefore,  that  much  of  the  immunity 
to  non-specific  poisons  is  dependent  on  the  functional  activity  of 
this  organ,  which  is,  so  to  speak,  interpolated  in  the  blood-current 
in  order  that  it  may  rid  the  circulation  of  certain  poisons,  and 
that  these  are  eliminated  in  the  bile  or  submitted  to  various 
chemical  modifications,  in  the  course  of  which  they  become  inert, 
or  at  least  less  toxic. 

Examples  of  the  neutralization  of  poisons  in  the  body  are 
numerous,  the  most  important  being,  perhaps,  the  formation  of 
urea,  a  relatively  non-toxic  substance,  by  the  synthesis  of  ammonia 
and  carbonic  acid,  followed  by  dehydration,  this  process  taking 
place  mainly  or  entirely  in  the  liver.  Other  syntheses,  however, 
take  place  in  other  organs — for  example,  the  union  of  glycocoll 
and  benzoic  acid  is  performed  in  the  kidney.  And  we  shall  see 
subsequently  that  the  leucocytes  and  leucocytic  organs  have  a 
very  special  and  important  duty  to  perform  in  dealing  with  poisons 
of  all  sorts,  both  organic  and  inorganic,  though  the  exact  chemical 
processes  that  they  bring  about  are  unknown.  It  will  be  more 
convenient  to  defer  the  consideration  of  these  cells  for  the 
present. 

The  exact  application  of  these  "non-specific"  processes  of 
detoxication  to  the  bacterial  toxins  has  not  yet  been  worked  out, 
but  there  can  be  little  doubt  of  its  great  importance.  Compare, 


IMMUNITY   TO   TOXINS  117 

for  example,  the  severity  of  the  symptoms  due  to  an  abscess  due 
to  B.  coli  and  the  lack  of  symptoms  due  to  the  absorption  of  the 
toxins  of  this  organism  which  occurs  from  the  alimentary  canal, 
and  very  probably  from  bacilli  which  actually  make  their  way 
through  its  walls  into  the  lacteals.  There  can  be  but  little  doubt 
that  the  symptoms  of  intoxication  in  the  former  case  are  due  to 
the  direct  access  of  the  bacterial  poisons  into  the  blood-stream 
without  having  first  to  traverse  the  liver.  Again,  in  a  case  of 
diphtheria  of  moderate  severity  virulent  bacilli  are  often  present 
in  abundance  so  great,  that  by  comparison  with  the  toxin-forming 
powers  of  the  organisms  in  vitro  we  might  expect  a  rapidly  fatal 
issue  from  the  toxin  absorbed,  and  yet  the  symptoms  of  general 
intoxication  may  not  be  severe.  In  all  probability  only  a  small 
fraction  of  the  toxin  formed  actually  reaches  the  distant  tissues. 
Some  may  be  dealt  with  by  the  zone  of  leucocytes  which  under- 
lies the  layer  of  diphtheria  bacilli,  but  it  is  also  possible  that  some 
of  the  toxin  is  destroyed  in  the  blood-stream,  perhaps  by  a  process 
of  oxidation.  These  non-specific  methods  of  dealing  with  toxins 
are  matters  more  for  the  physiologist  than  for  the  pathologist, 
depending  as  they  do  simply  on  the  perfect  discharge  of  the 
normal  functions  of  the  body.  They  are  of  great  importance — 
greater,  perhaps,  than  pathologists  usually  realize,  the  interest 
attaching  to  them  being  so  much  less  than  that  which  is  connected 
with  the  study  of  the  antitoxins  and  similar  bodies.  They  are  to 
be  regarded  as  the  first  line  of  defence  against  poisons  of  all  sorts. 
When  a  small  dose  of  a  specific  bacterial  toxin  gains  access  it  is 
probable  that  the  natural  physiological  methods  in  daily  use  for 
dealing  with  the  natural  poisons  (formed  in  metabolism  or  absorbed 
from  the  alimentary  canal)  are  in  most  cases  applicable  to  it  also, 
and  no  specific  process  has  to  be  brought  into  action.  In  confir- 
mation of  this  view  is  the  fact  that  diphtheria  antitoxin  is  not 
necessarily  found  in  the  blood  after  natural  recovery  from  diph- 
theria, suggesting  that  in  these  cases  the  processes  at  work  are 
non-specific. 

But  when  the  toxin  is  present  in  greater  amount  this  process 
may  fail,  and  it  may  do  so  in  consequence  of  the  action  of  the 
poison  on  the  tissues  and  organs  normally  concerned  in  the  defence 
of  the  body.  The  neutralization  of  toxins,  etc.,  in  the  liver  and 
other  regions  can  be  carried  out  best  when  these  organs  are  in  a 
state  of  high  functional  activity,  and  when  this  is  impaired  some 
of  the  deleterious  substances  will  escape  their  action.  Thus  we 


Il8  FORMS    OF    IMMUNITY   TO    TOXINS 

get  a  vicious  circle.  The  poison  injures  the  liver  or  other  organ 
and  impairs  its  defensive  powers.  More  poison  is  allowed  to 
circulate  in  the  blood,  and  the  liver  is  injured  still  more.  When 
this  occurs  we  are  thrown  back  on  the  specific  methods  of  dealing 
with  the  toxins,  which  take  longer  to  come  into  action,  and  which 
may  be  regarded  as  the  second  line  of  defence.  In  one  case, 
however,  we  must  regard  them  as  of  chief  importance.  Thus  in 
tetanus  the  toxin  is  produced  locally,  and  ascends  the  nerve - 
trunks  without  entering  the  blood-stream  or  being  carried  to  the 
liver  or  leucocytic  organs.  Here,  then,  in  all  probability,  specific 
methods  have  to  be  resorted  to,  and  the  chance  of  recovery  depends 
on  their  early  development. 

The  question  of  specific  immunity  to  toxins,  and  of  recovery 
from  intoxication  with  true  toxins,  may  be  considered  under  three 
heads : 

1.  Acquired  immunity,  due  to  disease  or  to  vaccination  with 
toxins  or  toxoids  (chemical  vaccination). 

2.  Antitoxic  or  passive  immunity,  due  to  the  injection  of  anti- 
toxic serum,  or  possibly  to  its  natural  occurrence  in  the  blood. 

3.  Natural  immunity,  not  due  to  vaccination,  and  not  accom- 
panied by  antitoxin  in  the  blood. 

It  will  be  convenient  to  consider  them  in  this  order. 

Natural  recovery  from  toxic  diseases  such  as  diphtheria  and 
tetanus  is  not  necessarily  accompanied  by  the  appearance  of  anti- 
toxin in  the  blood — at  least,  not  in  demonstrable  amounts.  Further, 
it  appears  from  the  researches  of  Abel  that  antitoxin,  when  it  is 
formed  at  all,  does  not  make  its  appearance  until  about  the  eighth 
day  of  the  disease,  at  which  period  the  severity  of  the  toxic 
symptoms  may  have  already  begun  to  decline.  What  may  be 
called  the  simple  antitoxic  theory  cannot  be  maintained.  Under 
natural  conditions  the  process  of  recovery  is  not  due  simply  to  the 
production  of  antitoxin,  though  it  is  possible  that  this  comes  into 
action  in  prolonged  cases. 

Further,  acquired  immunity  to  toxins  is  not  due  solely  to  the 
presence  of  antitoxin  in  the  blood.  If  it  were  so  it  should  develop 
proportionately  to  the  development  of  antitoxin,  and  the  two  should 
persist  for  the  same  length  of  time,  and  disappear  together.  This, 
however,  is  not  the  case.  We  have  already  seen  that  animals  in 
the  early  stage  of  immunization  to  diphtheria  and  tetanus  frequently 
present  a  decreased  amount  of  resistance,  or  hypersensitiveness, 
to  these  substances,  and  may  die  with  symptoms  of  acute  intoxica- 


IMMUNITY   TO   TOXINS  IIQ 

tion,  in  spite  of  the  presence  in  the  blood  of  amounts  of  antitoxin 
sufficient  to  neutralize  the  toxin  many  times  over.  Similar  appear- 
ances may  be  seen  in  animals  in  which  death  from  acute  intoxica- 
tion does  not  occur.  Thus,  in  two  horses  which  were  treated  at 
the  same  time  for  the  production  of  diphtheria  antitoxin,  one 
showed  but  little  sign  of  the  action  of  the  toxin,  and  improved  in 
general  health  during  the  treatment — it  developed  only  5  units  of 
antitoxin  per  cubic  centimetre  of  serum  ;  the  other  suffered  severely 
in  general  health,  and  had  ultimately  to  be  killed — it  developed 
70  units  per  cubic  centimetre.  Similar  phenomena  are  often  met 
with,  and,  as  a  general  rule  (to  which  there  are  numerous  excep- 
tions), the  presence  of  a  large  amount  of  antitoxin  in  the  blood 
indicates  susceptibility  rather  than  immunity.  The  facts  of  the 
later  stages  of  antitoxin-formation  may  also  be  borne  in  mind. 
Sooner  or  later  in  the  history  of  any  antitoxin  horse  there  comes  a 
time  when  the  amount  of  antitoxin  begins  to  diminish,  and  would, 
in  all  probability,  disappear  entirely  if  the  injections  were  con- 
tinued ;  yet  these  horses  are  extremely  resistant  to  the  action  of 
toxin — more  so,  in  fact,  than  animals  with  much  antitoxin  in  the 
blood.  The  degree  of  immunity,  therefore,  is  not  measured  by 
the  amount  of  antitoxin  in  the  blood,  and  we  might  argue  that  the 
two  are  not  related  in  any  way.  This,  however,  is  absurd,  in 
view  of  the  ascertained  action  of  antitoxin  in  neutralizing  the 
effects  of  toxin  in  vitro,  or  of  protecting  against  a  dose  surely  fatal. 
We  may  consider  the  role  of  antitoxin  under  two  heads  :  (i)  in 
immunity,  and  (2)  in  recovery  from  disease. 

Considering  first  the  question  why  an  animal  dies  in  spite  of  the 
presence  of  an  excess  of  antitoxin  in  the  blood,  we  must  regard 
the  simplest  and  most  probable  explanation  as  one  on  which  the 
toxin-antitoxin  molecule  is  looked  upon  as  dissociable.  This  is 
always  the  case  on  Arrhenius  and  Madsen's  theory  of  the  inter- 
action of  the  two  substances,  whilst  on  the  colloid  theory  the  dis- 
sociation only  takes  place  for  a  short  time  after  the  compound  has 
formed,  the  union  between  the  two  substances  gradually  becoming 
firmer  and  firmer.  On  this  supposition  the  failure  of  antitoxin  is 
readily  explicable  :  the  two  substances  unite,  and  the  inert  mole- 
cule is  formed  ;  this  dissociates,  and  the  toxin-molecules,  which 
happen  to  be  set  free  in  the  neighbourhood  of  susceptible  cells, 
unites  with  them.  The  removal  of  some  of  the  molecules  of  toxin 
allows  more  dissociation  to  take  place,  and  ultimately  the  whole  of 
this  substance  is  passed  on  to  the  tissues.  We  must  assume  that 


I2O  ROLE    OF   THE    LEUCOCYTES 

the  compound  formed  between  the  toxin  and  the  tissues  does  not 
dissociate,  as,  indeed,  appears  probable. 

But  if  this  is  the  case,  the  effect  of  antitoxin  in  the  blood  would 
be  merely  to  delay  the  action  of  the  toxin ;  assuming  this,  how 
are  we  to  explain  the  preventive  effect  of  this  substance  in  passive 
immunity  and  its  curative  effect  in  disease  ?  For  it  is  only  in  com- 
paratively rare  cases  in  the  early  stages  of  antitoxin  formation 
that  the  phenomena  under  discussion  occur,  and  in  all  other  con- 
ditions the  presence  of  a  sufficient  amount  of  antitoxin  in  the 
blood  constitutes  a  perfect  safeguard  against  the  action  of  its 
corresponding  toxin.  It  is  probable  that  the  leucocyte  is  the  all- 
important  factor  necessary  for  the  destruction  of  these  specific 
toxins,  whether  previously  neutralized  by  antitoxin  or  not.  We 
must  look  upon  the  toxin-antitoxin  molecule  as  one  which  can  be 
easily  ingested  and  destroyed  by  the  leucocytes,  and  the  neutraliza- 
tion of  toxin  by  antitoxin  as  the  first  step  in  a  double  process,  the 
second  being  the  destruction  of  the  compound  by  the  leucocytes. 
If  antitoxin  is  absent,  the  leucocytes  may  still  deal  successfully 
with  the  toxin,  if  the  latter  be  not  too  virulent,  nor  present  in  too 
large  an  amount ;  but  if  the  leucocytes  make  default,  the  presence 
of  antitoxin  may  delay  the  lethal  issue,  though  it  is  powerless  to 
avert  it.  We  may,  perhaps,  compare  antitoxin  with  opsonin, 
which  unites  with  bacteria  and  renders  them  suitable  for  ingestion 
by  the  white  corpuscles.  Metchnikoff  and  his  school  have  paid 
great  attention  to  the  role  of  the  leucocytes  in  intoxication,  and 
have  brought  forward  very  important  and  suggestive  evidence, 
pointing  to  the  white  corpuscles,  and  especially  the  large  lympho- 
cytes (macrophages)  as  the  main  source  of  antitoxin.  This 
question  is  dealt  with  elsewhere,  and  at  present  we  have  to  discuss 
only  the  role  of  the  leucocytes  in  dealing  with  the  toxins,  either 
alone  or  when  neutralized  by  antitoxin. 

The  evidence  proving  the  importance  of  the  leucocytes  in  deal- 
ing with  unaltered  toxins  is  abundant,  and  some  of  the  more  striking 
facts  brought  forward  by  the  French  school  have  been  briefly 
summarized  in  the  last  chapter.  The  evidence  for  the  removal 
of  toxin  in  combination  with  antitoxin  by  leucocytic  action  is  less 
direct,  since  it  is  obviously  impossible  to  recognize  this  compound 
by  chemical  or  microscopical  processes  whilst  in  the  leucocyte. 
But  it  has  been  shown  that  where  toxins  are  introduced  in  com- 
bination with  solid  substances,  such  as  carmine  or  brain  tissue, 
they  become  surrounded  by  large  numbers  of  leucocytes,  and  the 


IMMUNITY  TO   TOXINS  121 

particles  are  taken  up  by  these  cells.  Further,  the  injection  of 
toxin  into  an  animal  which  already  contains  antitoxin  in  the  blood 
brings  about  a  more  or  less  marked  leucocytosis.  It  is  true  that 
the  local  leucocytosis  brought  about  by  the  injection  of  neutral 
mixtures  of  the  two  may  be  but  slight,  but  we  must  remember 
that  substances  in  a  state  of  solution  are  quickly  absorbed  and 
carried  to  the  lymph  glands  or  bloodvessels,  in  either  of  which 
situations  leucocytes  occur  in  plenty.  Further,  there  are  always 
some  leucocytes  in  the  tissues,  and  the  number  may  be  sufficient 
to  deal  with  the  amount  of  toxin-antitoxin  injected,  which,  con- 
sidered as  mere  weight,  is  always  very  small. 

The  effect  of  antitoxin  on  the  toxin  of  B.  pyocyaneus  may  also  be 
cited  as  a  phenomenon  capable  of  explanation  on  this  supposition. 
A  few  lethal  doses  of  toxin  are  fully  neutralized  by  antitoxin,  the 
law  of  multiple  proportions  holding  up  to  a  certain  point.  When, 
however,  more  than  ten  lethal  doses  are  injected  the  law  does  not 
hold,  and  no  amount  of  antitoxin  will  avert  the  fatal  issue.  Here 
we  must  assume  that  the  antitoxin  has  but  a  slight  affinity  for  the 
toxin,  so  that  dissociation  takes  place  rapidly,  and  the  ultimate 
cause  of  the  destruction  of  the  toxin  to  be  leucocytic  activity. 
The  single  lethal  dose  is  just  more  than  the  leucocytes  can  deal 
with ;  but  when  antitoxin  is  injected  simultaneously  the  leucocytes 
have  time  to  come  into  action,  and  the  toxin,  gradually  set  free  by 
dissociation,  is  dealt  with  in  detail  instead  of  in  a  single  dose. 
There  are,  however,  limits  to  this  process,  and  when  more  than 
ten  lethal  doses  are  present  fully  combined  with  toxin  we  must 
imagine  that  dissociation  goes  on  so  rapidly  that  more  toxin  than 
one  lethal  dose  is  set  free  before  the  toxin-antitoxin  molecules  can 
be  taken  up.  If  this  explanation  is  highly  theoretical,  it  seems 
the  best  that  is  available  at  present. 

The  difference  between  the  effect  of  toxin  in  animals  with  anti- 
toxin in  the  blood,  but  otherwise  normal,  and  its  effect  in  animals 
in  the  early  stage  of  antitoxin  formation,  is  explicable  if  we  regard 
the  hypersensitiveness  as  occurring  in  the  leucocytes  themselves 
as  well  as  in  the  tissues.  When  antitoxin  is  injected  into  a  normal 
animal,  the  leucocytes  of  which  are  functionally  active,  the  con- 
ditions for  the  immediate  destruction  of  the  inert  molecule  are 
present,  and  no  dissociated  toxin  gains  access  to  the  susceptible 
tissues.  But  in  hypersensitive  animals  the  leucocytes  will  be 
unable  to  deal  with  the  molecule,  or  will  perhaps  be  killed  by  toxin 
liberated  by  dissociation  taking  place  within  their  protoplasm,  and 


122  ROLE    OF   THE    LEUCOCYTES 

the  toxin  will  ultimately  reach  the  tissues.  And  it  is  probable 
that  the  beneficial  effect  of  early  as  compared  with  late  injections 
of  diphtheria  toxin  may  be  dependent  in  part  on  the  fact  that  in 
this  disease  me  leucocytes  are  very  apt  to  suffer  degenerative 
changes  which  doubtless  impair  their  defensive  powers.  Ewing 
believes  that  the  variations  in  the  staining  capacity  of  these  cells 
(loss  of  chromatin  being  an  early  and  easily  recognized  sign  of 
their  degeneration)  might  be  utilized  as  a  means  of  prognosis,  and 
points  out  that  in  cases  which  recover  the  staining  quality  of  many 
of  the  leucocytes  undergoes  a  rapid  improvement  after  the  injec- 
tion of  antitoxin,  whereas  in  fatal  cases  this  change  could  not  be 
detected.  As  a  rule  the  total  number  of  leucocytes  is  diminished 
immediately  after  the  injection  of  antitoxin,  but  in  some  fatal 
cases  the  previously  existing  leucocytosis  may  remain  or  even 
increase.  This  fall  in  the  total  number  of  leucocytes  is  probably 
to  be  accounted  for  by  the  neutralization  of  the  toxin,  so  that 
fresh  white  corpuscles  are  no  longer  attracted  from  the  bone- 
marrow  by  positive  chemotaxis,  whilst  the  old  and  injured  cells 
remain  in  the  spleen  or  other  internal  organs,  no  longer  circulating 
in  the  blood.  In  cases  which  die  the  reduction  in  the  number  of 
leucocytes  may  be  followed  by  a  subsequent  rise. 

Here  also  we  must  refer  to  numerous  researches,  emanating 
more  especially  from  the  French  school,  which  go  to  show  that 
substances  which  are  certainly  devoid  of  true  specific  antagonism 
have  nevertheless  the  power  of  preventing  or  diminishing  the 
action  of  toxins  in  virtue  of  causing  an  increased  inflow  of  leuco- 
cytes to  the  region  into  which  the  injections  are  made.  This 
question  has  been  more  especially  investigated  in  regard  to  the 
effect  of  this  artificially  produced  local  leucocytosis  in  causing 
local  bacterial  immunity,  but  Calmette  and  Delecarde  have  shown 
that  the  peritoneal  injections  of  ordinary  broth  cause  a  certain 
degree  of  immunity  to  abrin,  whereas  normal  saline  solution  has 
no  such  power.  The  former  substance  is  a  powerful  agent  in 
attracting  leucocytes,  whilst  the  latter  is  much  less  potent  in  this 
respect,  though  by  no  means  devoid  of  activity.  Substances  which 
have  the  power  of  increasing  the  action  of  the  leucocytes  in  this 
way  are  sometimes  termed  "stimulins,"  but  the  word  should  be 
avoided.  The  action  of  leucocytes  in  any  region  may  be  in- 
creased by  many  methods,  the  chief  of  which  are :  (i)  attracting 
larger  numbers  by  means  of  substances  having  a  positive  chemo- 
tactic  action ;  (2)  stimulating  the  activity  of  the  leucocytes ;  and 


IMMUNITY   TO   TOXINS  123 

(3)  altering  the  nature  of  the  substances  on  which  they  are  to  act. 
If  the  term  "stimulin"  is  to  be  used  at  all,  it  should  be  rigidly 
restricted  to  one  that  acts  in  the  second  way,  and  there  is  as  yet 
no  strict  proof  that  such  a  body  occurs.  Substances  acting  in  the 
third  way  are  called  opsonins,  and,  if  the  theories  of  antitoxic 
action  here  outlined  are  correct,  antitoxins  have  this  action  also. 

Turning  now  to  the  process  of  recovery  from  bacterial  intoxi- 
cations in  natural  disease,  we  find  but  little  evidence  that  the 
formation  of  antitoxin  plays  any  active  part  in  the  process.  The 
conditions  are  not  as  a  rule  suitable  for  the  production  of  this 
substance,  since  the  constant  flow  of  the  toxin  into  the  circulation 
would  lead  rather  to  a  summation  of  negative  phases  and  the  pro- 
duction of  hypersensitiveness.  Further,  the  process  of  recovery 
may  have  commenced  before  antitoxin  appears  in  the  blood — a 
phenomenon  which,  when  it  occurs,  we  may  regard  rather  as  a 
means  of  preventing  reinfection  than  as  a  mechanism  for  the  cure 
of  the  original  attack  of  the  disease.  (It  may  be  pointed  out  that 
in  the  case  of  diphtheria  the  studies  of  Ruth  Tunnicliffe  lead  us  to 
believe  that  the  process  of  recovery  runs  parallel  to,  and  is  due 
to  a  rise  in,  the  opsonic  index,  and  that  the  main  factor  in  the  cure 
of  the  disease  is  the  removal  of  the  bacilli  by  a  process  of  phago- 
cytosis, and  consequent  cessation  of  the  absorption  of  toxin.)  In 
prolonged  diseases  it  is  possible  that  the  production,  and  especially, 
perhaps,  the  local  production  of  antitoxin,  may  play  some  part  in  the 
process.  In  other  cases,  perhaps,  the  toxin  is  dealt  with  by  some  of 
the  simpler  chemical  processes  considered  already  and  submitted  to 
various  changes  of  oxidation,  etc.,  and  thus  rendered  inert ;  but  of 
this  we  have  but  little  knowledge,  the  main  studies  of  the  methods 
in  which  the  body  deals  with  toxins  having  been  devoted  to  the 
phenomena  occurring  in  highly  immunized  animals  and  brought 
about  by  their  sera. 

Some  experiments  designed  by  Wassermann  as  evidence  in 
support  of  the  side-chain  theory  may  be  alluded  to  in  this  con- 
nection. He  found  that  the  injection  of  tetanus  toxoids  into 
highly  susceptible  animals  (guinea-pigs)  has  the  power  of  render- 
ing them  partially  immune  to  tetanus  toxin  for  a  short  period. 
Thus,  when  the  toxoid  was  injected,  followed  in  an  hour's  time 
by  unaltered  toxin,  the  lethal  dose  of  the  latter  was  greater  than 
in  a  normal  animal.  On  the  other  hand,  after  a  day  or  two  the 
animal  became  more  susceptible,  being  killed  by  less  than  the 
minimal  lethal  dose  for  normal  guinea-pigs.  This  he  explained 


124  RECOVERY    FROM    INTOXICATIONS 

by  supposing  that  the  receptors  of  the  sensitive  cells  were  occupied 
by  the  inert  toxoid,  so  that  part  of  the  cells  would  be  unable  to 
combine  with  the  true  toxin.  The  subsequent  susceptibility  he 
explains  by  the  over-production  of  new  receptors.  Now  when 
we  consider  that  in  acute  disease  of  natural  occurrence  the  pro- 
duction of  toxin  (in  cases  that  recover)  is  only  temporary,  and  is 
stopped  sooner  or  later  by  the  destruction  of  the  bacteria,  it  seems 
probable  that  some  such  process  may  occur  and  serve  to  shorten 
the  period  during  which  the  cells  are  exposed  to  the  action  of  the 
toxin.  The  true  toxins  are  so  fragile  that  it  is  highly  probable 
that  a  certain  proportion  of  them  are  converted  into  toxoids  during 
or  after  the  process  of  absorption  into  the  blood-stream,  and  by 
occupying  their  receptors  diminish  the  susceptibility  of  these  cells 
to  the  true  toxin,  which,  circulating  in  the  blood,  may  be  dealt 
with  by  the  leucocytes,  liver,  or  other  organs.  Afterwards  anti- 
toxin would  appear  in  the  blood,  but  it  would  have  no  relation  to 
the  early  slight  immunity  of  the  tissues. 

We  may  therefore  recognize  the  following  stages  in  the  process 
of  recovery  from  a  bacterial  intoxication  : 

1.  In  the  first  the  processes  are  mainly  non-specific,  the  chief 
being  probably  leucocytic  action  ;  the  dilution  of  the  toxin  in  the 
general  blood-stream,  and  its  elimination,  either  in  a  natural  con- 
dition or  after  it  has  undergone  some  chemical  alteration ;    the 
partial  destruction  and  conversion  of  toxin  into  toxoids,  and  sub- 
sequent  "  blocking "  of  side-chains  in  highly  important  tissues. 
In  mild  cases  these  may  be  sufficient  for  the  restoration  of  health, 
and  no  immunity,  or  the  merest  trace,  may  follow. 

2.  Where  the  process  lasts  longer  antitoxin  begins  to  be  pro- 
duced, and  in  all  probability  the  time  which  must  elapse  before 
this  occurs  varies  greatly  in  different  conditions,  depending  on : 
(a)  the  constitution  of  the  patient,  (b)  the  dose  of  the  toxin,  and 
(c)  on  the  nature  of  the  toxin.     Thus,  with  regard  to  the  last 
point,   it   may   be    remarked    that   it   appears   to   be   extremely 
difficult  to  prepare  antitoxins  to  the  endotoxins,  and  it  is  highly 
probable  that  in  recovery  from   such   diseases   as   cholera  and 
typhoid  fever  the   production   of   antitoxin  is   slight  or  absent. 
Such  diseases  owe  their  symptoms  mainly  to  the  production  of 
endotoxin,  and   are   combated    mainly   by   the   removal   of    the 
bacteria  which  produce  them.     In  the  cases  in  which  antitoxin  is 
produced,  recovery  at  this  stage  depends  on  its  presence,  together 
with  the  functional  efficiency  and  sufficient  numbers  of  the  leuco- 


IMMUNITY   TO   TOXINS  125 

cytes,  and  the  continuance  of  the  factors  by  which  the  toxin  is 
combated  in  the  first  stage  of  the  infection. 

Passive  antitoxic  immunity  consists  in  the  artificial  production 
of  this  stage. 

3.  In  the  third  stage,  which  is  only  reached  in  hyperimmunized 
animals  submitted  to  treatment  for  long  periods,  the  conditions 
are  quite  different,  and  do  not  appear  to  depend  in  any  way  on  the 
action  of  antitoxin,  but  approach  more  nearly  to  the  state  of 
natural  immunity  to  be  treated  subsequently.  In  this  condition  the 
antitoxin  in  the  blood  falls  greatly,  and  would  probably  disappear 
entirely  if  the  treatment  were  carried  further,  yet  the  degree  of 
immunity  is  very  great.  There  are  two  or  three  theories  which 
have  been  invoked  to  explain  this  form. 

In  the  first  place,  if  we  accept  the  side-chain  theory,  we  may 
ascribe  it  to  the  absence  of  receptors  suitable  for  the  toxin  which 
is  being  injected,  and  may  suppose  that  the  repeated  and  pro- 
longed stimulation  of  the  production  of  this  particular  receptor 
has  led  firstly  to  a  hypertrophy,  and  secondly  to  an  atrophy  of 
these  organs.  This  is  readily  conceivable,  and  might  be  compared 
with  the  sequence  of  hypertrophy  and  failure  of  the  heart  so 
frequently  met  with  in  Bright's  disease,  etc.  In  this  case  the 
toxin  would  be  unable  to  "  anchor  "  itself  to  the  cells,  which  would 
thus  escape  its  action,  and  the  result  would  be  one  form  of  tissue 
immunity.  There  is  but  little  experimental  evidence  bearing 
directly  on  this  theory  either  for  or  against.  The  state  is  one 
rarely  seen  even  under  experimental  conditions,  though  of  great 
theoretical  interest.  It  is  found,  however,  that  during  the  immuni- 
zation of  the  rabbit  with  eel  serum  (a  potent  haemolytic  agent)  an 
antitoxin  or  antihaemolysin  is  produced,  whereas  the  corpuscles 
themselves,  if  carefully  washed  from  all  traces  of  serum,  are  as 
susceptible  as  before.  If,  however,  the  injections  are  repeated, 
the  antitoxin  disappears,  and,  as  Tchistovitch  showed,  about  this 
time  the  red  corpuscles  themselves  become  immune,  not  being 
haemolyzed  by  eel  serum,  even  although  all  traces  of  their  own 
serum  is  washed  out.  This  we  may  fairly  suppose  to  be  due  to  a 
loss  of  the  receptors  with  which  the  molecules  of  eel  serum 
combine,  and  this  may  be  taken  as  an  experimental  demonstration 
of  the  occurrence  of  this  form  of  immunity,  which  was  predicted 
by  Ehrlich  on  theoretical  grounds.  Metchnikoff,  it  is  true, 
objects  to  this  hypothesis,  pointing  out  that  if  the  receptors  are  so 
necessary  for  the  nutrition  of  the  molecule  it  is  impossible  that 


126  TISSUE   IMMUNITY   TO   TOXINS 

these  latter  should  continue  to  live  after  the  receptors  had  been 
lost.  But  this  seems  based  on  a  misconception  of  Ehrlich's 
theories  of  cell-nutrition.  We  must  imagine  each  molecule  to  be 
provided  with  a  very  large  number  of  varieties  of  side-chain,  each 
adapted  to  the  seizure  of  a  different  form  of  food-molecule,  so 
that  the  complete  loss  of  one  variety  does  not  imply  that  the  cell 
will  suffer  in  nutrition.  The  only  result  is  that  more  work  will 
be  thrown  on  the  receptors  that  remain.  There  is  nothing  im- 
probable in  this.  It  is  in  the  highest  degree  likely  that  so 
important  a  function  as  nutrition  should  not  be  dependent  on  the 
functional  integrity  of  every  part  of  the  molecule.  The  occurrence 
of  tissue  immunity  from  loss  of  receptors  is  at  least  possible. 

An  alternative  theory  asserts  that  the  cells  of  the  body  retain 
their  power  of  combining  with  toxin,  but  lose  their  susceptibility 
to  the  action  of  the  toxophore  group  of  the  latter,  at  the  same  time 
losing  their  power  of  producing  antitoxin.  We  may  explain  the 
latter  phenomenon  by  saying  that  the  receptors  in  question 
become  sessile  and  incapable  of  being  shed.  There  is  experi- 
mental evidence  that  some  receptors  are  naturally  of  this  nature, 
and  it  is  quite  possible  that  ordinary  deciduous  receptors  might 
change  in  this  way.  As  we  shall  see,  tissue  immunity  in  which 
the  toxin  becomes  anchored  to  the  cell,  but  without  injuring  it  or 
stimulating  it  to  produce  antitoxin,  is  met  with  in  some  forms  of 
natural  immunity,  and  it  is  quite  possible  that  it  may  occur  in  the 
process  of  hyperimmunization  by  vaccines,  though  direct  proof  is 
lacking.  Another  process  of  somewhat  similar  nature  may  occur, 
which  has  also  its  analogue  in  natural  immunity.  Thus  when 
animals  are  immunized  to  tetanus  they  still  retain  their  suscepti- 
bility to  that  toxin  if  the  injection  be  made  directly  into  the 
brain.  It  is  obvious,  therefore,  that  the  cells  of  the  central 
nervous  system  are  still  susceptible.  Why,  then,  do  the  animals 
not  develop  tetanus  if  the  injections  be  made  subcutaneously  ? 
We  exclude,  of  course,  the  case  in  which  antitoxin  is  present  in 
the  blood  and  the  leucocytes  are  functionally  active.  It  will  be 
shown  subsequently  that  some  forms  of  natural  immunity  depend 
upon  the  fact  that  some  cells  in  the  body  unite  with  the  toxin,  and 
have,  indeed,  a  great  affinity  for  it,  but  are  not  injured  thereby, 
whereas  others  have  an  affinity  for  it,  but  are  killed  by  its  action. 
Obviously,  if  the  toxin  be  injected  into  tissues  of  the  former  class 
it  will  be  absorbed,  none  will  reach  the  susceptible  second  class  of 
cells,  and  no  toxic  symptoms  will  result.  Now  let  us  suppose 


IMMUNITY    TO   TOXINS  127 

that  in  immunizing  a  susceptible  animal  (rabbit  or  guinea-pig) 
to  tetanus  the  connective  tissues  acquire  receptors  suitable  for 
the  toxin  in  question.  They  will  absorb  this  toxin,  be  shed,  and 
the  animal  will  develop  antitoxin,  whilst  the  tissues  previously 
susceptible — the  brain  and  cord — will  remain  as  susceptible  as 
before.  This  is  probably  what  occurs,  though  the  critical  proof — 
the  demonstration  of  an  increase  in  the  toxin-absorbing  power  of 
the  tissues  in  acquired  immunity,  and  thus  of  an  increased  number 
of  receptors — does  not  appear  to  be  forthcoming.  Dmitrevsky 
investigated  the  amount  of  tetanus  toxin  neutralized  by  brain 
tissue  before  and  after  immunization ;  but  since,  as  we  have  seen, 
this  process  is  probably  different  in  nature  from  the  neutralization 
of  toxin  by  antitoxin,  his  researches  have  not  much  weight  in  this 
connection. 

The  third  explanation  is  that  of  Metchnikoff,  that  the  leucocytes 
themselves  have  become  immune  to  the  toxin,  and  have  thus 
acquired  the  power  of  dealing  with  that  substance  in  large 
quantities,  so  that  the  tissues  are  sheltered  from  its  action.  This 
theory  simply  shifts  the  immunity  back  to  the  leucocytes,  and 
thus  does  not  really  solve  the  problem,  which,  it  must  be 
admitted,  is  unsolved  whatever  theory  we  may  adopt,  and  perhaps 
insoluble  in  the  present  state  of  our  knowledge  of  the  physical 
chemistry  of  living  matter.  We  may,  however,  combine  it  with 
perfect  propriety  with  the  side-chain  theory,  and  argue  that  the 
leucocytes  may  have  behaved  in  the  same  way  as  we  have 
supposed  the  tissue  cells  to  have  done  in  the  last  paragraph — that 
is,  to  have  retained,  and  perhaps  increased,  their  receptors,  whilst 
losing  their  susceptibility  to  the  toxophore  group.  Here,  again, 
we  are  confronted  with  the  absence  of  any  definite  evidence  either 
way.  There  seems  to  be  no  experimental  proof  to  show  whether 
leucocytes  from  a  hypervaccinated  animal  have  any  increased 
resistance  to  the  poisonous  action  of  toxins  or  any  increased 
power  of  combining  with  them.  This  is  greatly  to  be  deplored, 
and  might  easily  be  remedied  now  that  Sir  Almroth  Wright  has 
taught  us  a  simple  and  convenient  method  of  working  with  living 
leucocytes.  As  far  as  our  experimental  knowledge  goes,  there  is  no 
great  difference  in  leucocytes  from  immunized  and  normal  animals, 
and  the  researches  on  opsonins  have  led  to  a  tendency  to  disregard 
the  possible  variations  in  the  immunity  of  the  leucocytes,  since 
the  researches  of  Bulloch  and  others  showed  that  leucocytes  from 
very  varied  sources  would  take  up  the  same  number  of  bacteria 


128  IMMUNIZATION    OF  LEUCOCYTES 

under  identical  conditions.  Subsequently,  as  we  have  seen  above, 
some  slight  indications  of  a  difference  have  been  found.  But  on 
Metchnikoff's  theories  there  ought  to  be  a  very  marked  difference, 
and  we  should  expect  that,  the  leucocyte  being  par  excellence  the 
cell  devoted  to  the  protection  of  the  animal  body  against  infections, 
it  would  be  the  first  to  acquire  increased  resistance  in  acquired 
immunity,  and  we  should  expect,  e.g.,  leucocytes  from  a  convales- 
cent case  of  pneumonia  or  from  an  animal  vaccinated  against  the 
pneumococcus  to  take  up  far  more  pneumococci  than  leucocytes 
from  a  normal  person  under  similar  conditions,  whereas  the 
difference,  if  one  exists,  is  extremely  slight.  A  careful  considera- 
tion of  the  conditions  of  opsonic  experiments  leads  to  the  con- 
clusion that  the  results  obtained  are  not  to  be  regarded  in  any 
sense  as  an  index  of  the  immunity  of  the  leucocytes.  Ledingham 
has  shown  that  the  number  of  bacteria  taken  up  by  the  leucocytes 
depends  on  the  extent  to  which  the  former  have  been  altered  by 
the  action  of  serum,  and  not  on  the  temperature  at  which  the 
phagocytosis  takes  place.  Thus,  if  the  bacteria  are  sensitized  by 
serum,  mixed  with  leucocytes,  and  part  kept  at  18°  C.  and  part  at 

-£*- c*^^«.  ^^esoX<_tt. 

37  C.,  the  number  of  leucocytes  is  the  same  in  the  two  cases. 
Now  at  the  lower  temperature  no  active  movements  of  the 
leucocytes  take  place,  so  that  we  cannot  regard  the  ingestion  of 
the  bacteria  as  being  entirely,  as  was  formerly  assumed,  an  active 
process  brought  about  by  a  seizure  or  surrounding  of  the 
organism  by  pseudopodia,  as  can  be  seen  so  readily  in  the 
amoeba.  Such  a  process  does  occur  to  some  extent,  and  can  be 
seen  to  occur  under  suitable  conditions  ;  but  it  is  also  true  that 
bacteria  can  be  seen  to  make  their  way  into  a  cell  which  has 
been  watched  continuously  under  a  high  power  of  the  microscope, 
and  in  which  no  movement  of  any  sort  has  been  witnessed.  We 
are  led,  therefore,  to  believe  that  the  phagocytosis  which  occurs 
in  opsonin  experiments  in  vitro  is  a  process  allied  to  agglutination 
rather  than  to  an  actual  physical  seizing  of  an  organism.  It  is 
one  which  can  take  place  without  an  actual  conscious — if  we  may 
use  the  term — movement  of  the  leucocyte  towards  its  prey. 
An  immunized  leucocyte  would  be  immune  to  bacteria  which  it 
had  ingested,  to  the  endotoxins  liberated  in  the  process  of 
bacteriolysis,  whether  taking  place  within  the  leucocyte  or  outside 
it,  and  to  endotoxins  ;  and  it  would  exert  its  physiological  functions 
of  movement  (pseudopodia- production  and  chemotaxis)  equally 
well  whether  these  toxins  were  present  or  absent.  Or  the  effect 


IMMUNITY   TO   TOXINS  I2Q 

of  a  given  toxin  might  be  reversed  in  the  immunized  leucocytes, 
so  that  in  place  of  being  repelled,  as  under  normal  conditions,  it 
might  be  attracted.  As  far  as  phagocytosis  depends  on  the 
chemotactic  attraction  of  leucocytes  and  the  active  suggestion  of 
organisms,  we  might  expect  it  to  be  greatly  increased  if  these 
cells  became  immune.  But  there  is  no  reason  to  see  how  a 
similar  increase  need  occur  if  it  depends  on  a  process  akin  to 
agglutination,  and  resulting,  perhaps,  from  a  change  in  the  surface 
tension  between  the  leucocyte  and  the  organism.  And  that  this  is 
the  way  in  which  the  vast  majority  of  the  bacteria  are  taken  up 
in  ordinary  opsonin  preparations  appears  probable.  Active  move- 
ments of  leucocytes  are  rarely  seen  in  emulsions  prepared  for  the 
opsonic  technique  and  examined  in  normal  saline  solution  on  the 
warm  stage.  In  other  words,  the  opsonic  index,  though  useful  as 
an  index  of  certain  activities  of  the  serum,  would  appear  not  to 
throw  much,  if  any,  light  on  the  immunity  of  the  leucocytes. 
That  must  be  sought  by  other  means,  notably  by  experiments  in 
vivo,  and  these  lead  us  to  the  belief  that  the  leucocytes  may 
become  immune  and  may  play  a  part  of  importance  in  acquired 
immunity. 

But  the  facts  which  have  been  previously  recorded  concerning 
the  local  reaction  in  animals  which  are  being  immunized  to  tetanus 
toxin  tend  to  support  MetchnikofFs  hypothesis,  though  the  evi- 
dence is  somewhat  indirect.  We  have  already  pointed  out  that 
when  normal  horses  are  injected  with  small  doses  of  this  toxin 
there  is  at  first  no  local  reaction,  or  but  slight,  and  that  as  the 
animal  becomes  more  immune  this  local  reaction  becomes  more 
manifest — a  phenomenon  which  we  have  attributed  to  the 
hypersensitiveness  of  the  tissues.  Now  the  local  reaction  is 
inflammatory  in  nature,  and  the  tumefaction  which  occurs  is  due 
partly  to  oedema  and  partly  to  an  access  of  leucocytes,  so  that  it 
would  appear  that  in  the  production  of  immunity  these  cells 
acquired  the  power  of  invading  a  tissue  which  is  permeated  with 
toxin..  If  this  is  the  case,  we  may  assume  that  they  have  become 
immunized  to  this  toxin,  and  that  they  have  changed  in  regard  to 
their  chemotactic  properties.  This  collection  of  leucocytes  in 
large  numbers  at  the  region  of  inoculation  of  toxins,  where  they 
may  form  a  sterile  abscess  of  considerableextent,  is  a  frequent 
phenomenon  in  the  production  of  diphtheria  toxin  ;  and  here,  again, 
it  seems  not  improbable  that  the  leucocytes  have  become,  like  the 
tissue  cells,  more  immune  to  the  toxin.  There  are  no  inherent 

9 


130  BIOLOGICAL   SIGNIFICANCE   OF   ANTITOXIN 

difficulties    in    the    supposition,    and    it    certainly   favours    the 
explanation  of  the  phenomenon. 

It  will  be  remarked  that  the  tendency  of  modern  thought  has 
been  rather  to  minimize  the  importance  of  antitoxin  in  the  process 
of  natural  recovery  and  in  the  subsequent  immunity,  and  to  regard 
its  appearance  rather  as  an  epiphenomenon,  though  doubtless  one 
that  may  under  certain  circumstances  be  of  advantage  to  the 
patient.  Let  us  consider  briefly  the  probable  significance  of  anti- 
toxin-formation in  the  animal  economy.  Is  it  a  purposive  pheno- 
menon, developed  and  selected  by  natural  selection  with  a  view  to 
defend  the  species  against  natural  dangers  ?  This  was  the  natural 
assumption  when  antitoxin  was  thought  to  play  a  role  of  the 
utmost  importance  in  natural  recovery  and  acquired  immunity. 
There  are  great  difficulties  in  the  way  of  accepting  such  a  hypo- 
thesis. In  the  first  place,  immunity  to  bacterial  infections  is  more 
prevalent  in  the  lower  rather  than  in  the  higher  animal  types. 
Doubtless  very  great  susceptibility  to  a  widespread  infective 
agent  would  operate  unfavourably  to  the  prospect  of  survival  of 
any  animal  species,  but  actual  experience  seems  to  show  that 
immunity  to  ordinary  infections  is  a  far  less  potent  factor  in 
evolution  than  those  studied  by  Darwin  and  his  school.  It  would 
appear  in  the  case  of  tetanus  at  least  that  susceptibility  is  acquired 
as  the  animal  rises  in  the  zoological  scale  as  a  secondary,  though 
perhaps  necessary,  result  of  the  possession  of  a  nervous  system  of 
a  certain  degree  of  complexity.  Secondly,  the  immunity  due  to  a 
successful  struggle  against  a  disease  is  an  acquired  factor,  and  as 
such  not  transmitted,  according,  at  least,  to  the  majority  of  modern 
authorities.  It  might  be  objected  that  the  capability  of  producing 
antitoxin  might  be  a  spontaneous  feature,  and  one  capable  of  being 
transmitted  by  heredity.  This  is  probably  true,  but  in  this  case  it 
could  only  become  a  powerful  agent  in  evolution  if  the  disease 
were  prevalent  and  an  attack  usual  in  the  life-history  of  many  of 
the  individuals.  This  might  occur  in  the  case  of  tetanus,  though 
here,  as  we  have  seen,  the  tendency  in  evolution  is  for  the  production 
of  susceptibility  rather  than  immunity  ;  but  it  is  quite  impossible 
to  see  how  the  power  of  forming  an  antitoxin  to  eel  serum  after 
the  injection  of  that  substance  (to  take  one  example  of  many 
which  might  be  quoted)  can  be  of  advantage  to  any  animal  unless 
its  habitat  happens  to  be  a  pathological  laboratory.  Lastly,  if  the 
acquirement  of  immunity  be  of  great  importance  in  natural 
selection,  we  should  expect  those  species  to  survive  which  de- 


IMMUNITY   TO   TOXINS  131 

veloped  natural,  rather  than  the  power  to  develop  acquired, 
immunity,  since  the  former  would  be  always  available,  whilst  the 
latter  only  come  into  action  after  the  animal  has  successfully  sur- 
mounted the  hazard  of  an  attack  of  the  disease. 

It  appears  more  likely  that  the  power  to  produce  antibodies 
under  certain  circumstances  is  to  be  regarded  as  one  of  the 
essential  properties  of  some  forms  of  living  protoplasm,  and  that 
its  occasional  value  in  the  cure  or  prevention  of  disease  is  a  mere 
accident.  Thus  the  injection  of  eel  serum  leads  to  the  production 
of  antitoxin  in  all  mammals,  as  far  as  is  known,  and  this  could 
only  be  of  advantage  to  the  animal  if  (i)  eel  serum  happened  to 
gain  access  to  the  tissues  ;  (2)  the  animal  recovered ;  and  (3)  eel 
serum  again  reached  the  tissues  within  a  certain  period.  This  is 
extremely  unlikely  to  occur.  Nor  is  natural  immunity  from 
poisons  necessarily  dependent  on  antitoxin ;  in  fact,  the  common 
vegetable  and  other  poisons  do  not  lead  to  a  production  of  anti- 
bodies, though  if  they  did  the  possession  of  these  substances 
would  doubtless  be  of  great  value  to  animals  in  a  wild  state.  If 
there  is  any  advantage  attaching  to  the  power  of  forming  anti- 
bodies, it  is  probably  in  regard  to  the  solution  of  organized  bodies, 
such  as  bacteria,  or  their  sensitization  previously  to  phagocytosis. 
It  is  quite  conceivable  that  the  advantage  accruing  from  the 
possession  of  the  power  of  forming  antibodies  has  led  to  the 
selection  of  animals  whose  protoplasm  has  the  property  of  develop- 
ing an  antibody  to  any  foreign  proteid,  the  useful  side  of  this 
property  being  the  formation  of  bacteriolysins  and  opsonins, 
the  useless  corollary  being  the  production  of  antitoxins  and 
precipitins. 

The  theory  of  passive  antitoxic  immunity  does  not  present  any 
difficulties  of  importance.  It  may  be  pointed  out  that  it  comes  on 
as  soon  as  the  antitoxin  reaches  the  blood-stream — i.e.,  at  once  if 
the  injection  be  intravenous,  and  after  a  delay  of  some  duration  if 
it  be  into  the  subcutaneous  tissues  or  peritoneum. 

Much  attention  has  been  paid  to  the  question  of  the  feasibility 
of  administering  antitoxin  by  the  mouth  and  rectum.  In  general 
there  is  no  doubt  that  this  is  useless,  and  under  ordinary  conditions 
it  is  not  absorbed  as  such,  being  probably  digested,  and  thus 
deprived  of  its  peculiar  characters.  Under  certain  circumstances 
this  appears  not  to  be  the  case ;  thus  it  is  held  that  absorption 
from  the  stomach  may  take  place  in  young  animals.  Hence, 
perhaps,  some  of  the  undoubted  benefit  of  the  use  of  fresh  raw 

9—2 


132  PASSIVE    IMMUNITY 

milk  (in  which  the  antibodies  have  not  been  destroyed  by  heat)  in 
the  feeding  of  infants.  It  seems  also  that  absorption  of  antitoxins 
can  take  place  in  the  intestine,  and  that  the  process  may  occur 
when  gastric  digestion  is  impaired  or  is  prevented  by  artificial 
means ;  thus  McClintock  and  King  obtained  successful  results  (in 
animals)  in  93  per  cent,  of  cases  after  the  administration  of  a 
mixture  of  diphtheria  antitoxin,  opium,  and  of  a  saturated  solution 
of  salol  in  chloroform.  It  is  possible  that  something  of  the  same 
sort  may  occur  in  disease  where  the  digestive  powers  are  notably 
enfeebled,  but  this  does  not  justify  its  exhibition  in  this  way  in 
cases  of  disease  where  every  hour  is  of  the  utmost  value.  Recent 
researches  have  also  shown  that  in  some  cases  animals  may  be 
immunized  per  rectum  ;  possibly  success  is  only  attained  when  the 
antitoxin  goes  sufficiently  high  up. 

As  regards  the  duration  of  the  immunity  conferred  by  a  single 
dose  of  antitoxin,  our  knowledge  is  not  very  exact  in  the  case  of 
human  beings.  According  to  von  Behring,  Goodman,  and  others,  an 
animal  injected  with  diphtheria  antitoxin  retains  its  immunity  for 
rather  more  than  three  weeks,  and  the  duration  is  not  markedly 
affected  by  the  size  of  the  dose  given — for  example,  20  units  of 
antitoxin  per  kilogramme  immunized  a  rabbit  twenty-three  days, 
500  units  twenty-six  days.  According  to  Goodman,  the  degree  of 
immunity  falls  rapidly  for  the  first  two  or  three  days,  and  then  more 
slowly.  According  to  him  also,  the  immunity  is  greater  than  the 
amount  of  antitoxin  in  the  blood  would  lead  one  to  suppose,  and  it 
continues  when  antitoxin  is  no  longer  demonstrable  in  that  situation. 
The  duration  of  the  immunity  appears  to  depend  upon  the  source  of 
the  antitoxin  injected,  being  longer  if  the  serum  is  homologous — 
i.e.,  derived  from  an  animal  of  the  same  species  :  thus  von  Behring 
showed  that  horses  immunized  by  horse-serum  antitoxin  retain 
their  passive  immunity  almost  as  long  as  if  they  had  acquired 
active  immunity  from  toxin  injections,  whereas  the  effect  in 
rabbits,  etc.,  as  we  have  seen,  is  more  transient. 

But  little  need  be  added  to  what  has  been  already  mentioned 
incidentally  with  regard  to  the  curative  power  of  antitoxin.  It 
acts,  in  the  main,  by  neutralizing  all  toxin  present  in  a  free  state 
in  the  blood  or  subsequently  formed  by  the  bacteria.  In  general, 
as  has  been  pointed  out,  it  does  not  repair  the  damage  that  the 
toxin  has  already  done,  nor  remove  the  latter  from  its  combination 
with  the  susceptible  cells.  It  is  perhaps  hardly  safe  to  assume  that 
there  is  no  trace  of  such  an  action,  and  it  is  by  no  means  improb- 


IMMUNITY   TO   TOXINS  133 

able  that  a  very  large  excess  of  antitoxin  present  in  the  blood  may 
have  some  slight  power  of  attracting  toxin  from  the  tissues  when 
the  union  is  but  recent.  This,  however,  is  quite  unproved,  but  it 
is  certain  that  cells  which  have  been  acted  on  by  the  toxin  are  not 
benefited  by  the  subsequent  action  of  the  antidote. 

It  is  hardly  necessary  to  point  out  that  antitoxin  is  devoid  of 
bactericidal  action,  and  that  the  removal  of  the  infective  agent  is 
brought  about  by  other  mechanisms — in  most  cases,  perhaps,  by 
phagocytosis.  Thus  we  shall  see,  in  the  case  of  diphtheria,  that 
there  is  a  rapid  rise  in  the  opsonic  index  aj^out  the  time  of  the 
improvement  in  the  local  lesions ;  and  we  can  hardly  doubt,  though 
direct  evidence  is  lacking,  that  the  preservation  of  the  leucocytes 
from  the  deleterious  action  of  the  toxin  when  the  latter  is  neutral- 
ized by  means  of  antitoxin  also  plays  its  part  in  facilitating 
phagocytosis. 

We  now  turn  to  the  subject  of  natural  immunity,  an  exceedingly 
difficult  one,  and  one  that  is  very  far  from  being  understood.  What 
follows  is  for  the  most  part  extremely  hypothetical. 

It  must  be  borne  in  mind  that,  though  we  speak  of  animals  as 
susceptible  or  as  immune  to  a  given  toxin,  no  hard-and-fast  line 
can  be  drawn  between  the  two  conditions,  and  in  some  cases  an  un- 
broken series  can  be  constructed  between  the  most  susceptible  and 
the  most  resistant  species.  This  is  well  seen  in  the  case  of  tetanus. 
According  to  Knorr,  the  horse  is  the  most  susceptible  animal 
to  the  toxin  of  this  disease  ;  the  guinea-pig  requires  twice  as  much 
toxin  per  kilogramme  of  body- weight  to  constitute  a  lethal  dose, 
the  goat  4  times  as  much,  the  mouse  13  times,  the  rabbit  2,000 
times,  and  the  hen  200,000  times.  Von  Behring  confirms  these 
figures  in  general,  but  finds  the  mouse  rather  more  susceptible 
than  the  horse.  With  the  exception  of  the  fowl  these  animals  are 
all  "  susceptible."  Of  the  "  insusceptible  "  animals  the  hen  is  sus- 
ceptible to  large  doses,  but  is  also  affected  by  much  smaller  ones 
if  the  injection  be  intra-cerebral.  Other  animals  are  still  more 
resistant,  such  as  the  tortoise,  lizard,  cayman,  larva  of  Oryctes, 
etc.  The  effect  of  other  toxins  on  different  species  of  animals 
has  not  yet  been  fully  investigated. 

It  is  necessary  to  realize  that,  before  we  can  say  that  an  animal 
is  insusceptible  or  immune  to  the  action  of  a  given  toxin,  it  is 
necessary  for  the  animal  to  be  kept,  after  injection,  at  a  tempera- 
ture at  which  this  toxin  can  act.  This  subject  has  already  been 
mentioned  in  connection  with  the  functions  of  the  haptophore  and 


134  NATURAL    IMMUNITY   TO   TOXINS 

toxophore  groups.  We  have  already  seen  reason  to  believe  that 
the  former  can  functionate  (i.e.,  the  molecule  of  toxin  can  unite 
with  the  cell)  at  a  low  temperature,  whereas  the  enzyme-like 
action  of  the  toxophore  radical  can  only  act  at  the  temperature  of 
the  human  body,  or  one  approximating  thereto — this  is  in  the  case 
of  tetanus.  It  is  apparent,  therefore,  at  the  outset  that  there  are 
two  possible  explanations  for  immunity  to  toxins :  firstly,  the 
toxin  may  find  no  cell  with  which  it  can  unite — or,  to  use  Ehrlich's 
terminology,  no  cell  receptor  having  a  combining  affinity  for  the 
haptophore  molecule  of  the  toxin  molecule ;  and,  secondly,  the 
union  takes  place,  but  the  cell  is  not  injured  as  a  consequence — 
that  is,  the  toxophore  group  is  without  action  on  the  cell  proto- 
plasm. 

1.  Immunity  due  to  an  absence  of  suitable  receptors. 

This  has  been  proved  to  exist  in  several  cases.  Most  of  these 
are  due  to  the  researches  of  Metchnikoff,  who,  however,  does  not 
express  the  fact  in  these  words,  but  contents  himself  by  saying 
that  the  toxin  does  not  combine  with  the  tissues.  Thus,  when  the 
larva  of  Oryctes  is  injected  with  tetanus  toxin,  no  symptoms 
develop,  and  if  the  blood  of  the  animal  be  collected  several  months 
afterwards,  it  will  be  found  to  contain  free  toxin,  as  shown  by  the 
fact  that  it  will  tetanize  susceptible  animals.  These  experiments 
were  carried  out  at  a  temperature  of  30°  to  36°  C.  Similar  facts 
were  observed  in  lizards ;  -^  c.c.  of  blood  taken  two  months  after 
the  injection  of  tetanus  toxin  produced  fatal  tetanus  in  a  mouse ; 
the  blood  of  a  turtle  (Emys  orUcularis)  contained  toxin  no  less 
than  four  months  after  injection.  In  these  cases  the  explanation  of 
the  immunity  is  clear,  however  we  choose  to  express  it :  the  toxin 
has  no  chemical  affinity  for  the  living  protoplasm  of  any  part  of 
the  animal,  and  is,  therefore,  powerless  to  injure  it. 

2.  Immunity  due   to   the   insusceptibility  of   the  cells  to  the 
action  of  the  toxophore  group. 

Perhaps  the  best  example  of  this  is  in  the  case,  so  often  referred 
to,  of  the  action  of  tetanus  toxin  on  the  cold  frog,  but  other 
striking  examples  have  been  discovered  by  Metchnikoff.  Thus, 
when  the  alligator  is  kept  at  ordinary  temperatures  (about  20°  C.) 
and  injected  with  tetanus  toxin,  that  substance  rapidly  disappears 
from  the  blood,  yet  without  the  production  of  any  tetanic  symptoms, 
and  without  the  appearance  of  antitoxin  in  the  blood.  If,  how- 
ever, the  animal  is  kept  at  a  temperature  of  32°  to  37°  C.,  it  pro- 
duces antitoxin  with  great  rapidity,  though  still  without  the 


IMMUNITY  To  TOXINS  135 

production  of  tetanus.  Taking  these  two  experiments  together, 
we  may  regard  them  as  constituting  a  definite  proof  of  the  existence 
of  this  form  of  immunity.  The  theory  that  the  toxin  disappears 
because  it  is  anchored  to  the  cells  appears  to  be  clearly  demon- 
strated from  the  fact  that  antitoxin  is  produced,  an  occurrence 
which  we  could  scarcely  explain  on  any  other  hypothesis. 

But  it  is  hardly  necessary  to  look  for  experimental  proof  of  the 
occurrence  of  this  form  of  immunity,  since  the  fact  that  the  blood 
of  many  species  of  animals  is  toxic  for  other  species  appears  to 
constitute  a  ready-made  demonstration  of  the  fact.  The  most 
striking  example  is  given  by  eel  serum,  a  substance  which  is  toxic 
for  nearly  all  animals,  except,  of  course,  the  eel  itself.  The  serum 
of  the  horse  is  perhaps  the  least  toxic  of  all  sera,  but  even  this 
has  some  poisonous  properties.  Whatever  theory  we  may  hold 
with  regard  to  the  mechanism  by  which  the  cells  of  an  animal 
are  nourished,  it  is  hardly  possible  to  avoid  the  conclusion  that  the 
proteid  substances  of  the  plasma  must  combine  with  the  living 
protoplasm.  And  it  is  the  proteids  which  make  eel  serum  toxic 
to  other  animals.  It  appears,  therefore,  that  the  immunity  of  the 
eel  to  its  own  serum  depends  upon  the  fact  that  its  protoplasm  is 
not  injured  by  the  toxophore  group,  although  the  molecules  of  the 
toxin  and  protoplasm  become  united.  (The  toxicity  of  eel  serum 
is  dependent  on  a  more  complex  structure  than  that  of  ordinary 
bacterial  toxins,  being,  in  fact,  a  cytolysin  or  cytotoxin,  but  this 
does  not  affect  the  argument.) 

A  consideration  of  the  use  of  these  poisonous  sera  to  the 
animals  which  produce  them  may  perhaps  enable  us  to  analyze 
the  process  a  step  further,  and  to  attribute  their  immunity  (e.g., 
of  eels  to  eel  serum)  to  the  power  which  their  cells  possess  of 
using  these  "  toxic  "  substances  as  sources  of  nourishment ;  and 
if  this  be  so,  we  might  perhaps  apply  this  suggestion  to  explain 
the  form  of  immunity  to  toxins  under  discussion.  Thus,  in  the 
case  of  the  alligator  injected  with  tetanus  toxin  and  kept  in  the 
cold,  it  is  possible  that  the  poison  which  combines  with  the  cells 
is  "  digested  "  by  them  and  used  as  nourishment.  If  this  is  so,  we 
must  assume  that  the  molecule  of  toxin  has,  as  far  as  its  hapto- 
phore  group  is  concerned,  a  close  chemical  affinity  with  the 
proteids  normally  present  in  the  animal's  blood  and  used  by  it  in 
cell  nutrition ;  and,  further,  that  the  toxophore  group  is  not  only 
innocuous  to  the  cell,  but  is  also  no  bar  to  the  assimilation  of  the 
whole  molecule  by  it.  We  have  seen  that  in  the  case  of  the  frog 


136  NATURAL   IMMUNITY   TO   TOXINS 

the  toxophore  group  only  acts  at  a  raised  temperature.  In  the 
alligator  we  must  assume  that  the  elevation  of  the  temperature 
has  a  similar  but  less  marked  effect ;  it  brings  the  toxophore  group 
into  a  state  of  activity  in  which  it  is  inassimilable  by  the  molecule 
of  cell  protoplasm  to  which  it  is  anchored,  although  the  latter  is 
not  injured  in  any  way.  The  result  is,  of  course,  the  production 
of  antitoxin,  which  approximates  to  the  formation  of  the  pre- 
cipitins  which  occurs  when  non-poisonous  foreign  proteids  are 
injected. 

Thus  it  would  appear  that  at  the  last  analysis  this  form  of 
immunity  is  in  reality  an  expression  of  cell  nutrition.  A  cell 
which  can  nourish  itself  on  a  given  toxin  is  naturally  immune  to 
its  action.  It  is  hardly  necessary,  however,  to  say  that  there  is 
no  direct  proof  that  any  cell  can  extract  nourishment  from  a 
bacterial  toxin. 

There  is  a  third,  and  in  some  respects  more  interesting  and 
important,  form  of  immunity  to  toxins  which  is  readily  conceivable 
on  theoretical  grounds,  and  the  occurrence  of  which  is  capable  of 
experimental  proof.  We  may  define  it  as  immunity  due  to  the 
fact  that  some  of  the  cells  are  insusceptible  to  the  action  of  the 
toxin,  but  have  a  great  combining  affinity  therewith,  and  thus 
shield  from  its  action  the  susceptible  cells,  in  which  the  combining 
affinity  is  less.  We  have  already  referred  to  the  most  striking 
and  best  known  example,  that  of  the  immunity  of  the  fowl  to 
tetanus  toxin  injected  subcutaneously  as  compared  with  its  sus- 
ceptibility when  the  injection  is  made  direct  into  the  brain.  Some 
authorities  have  attempted  to  explain  this  on  the  assumption  that 
there^is  a  barrier  en  route  which  prevents  the  access  of  the  toxin 
to  the  central  nervous  system.  But  if  by  this  we  are  to  imagine  a 
physical  barrier  in  the  shape  of  a  layer  of  endothelium  or  other 
tissue  between  the  blood  and  the  cells  of  the  brain,  the  explanation 
is  inadequate,  since  it  would  leave  unexplained  the  nature  of  the 
immunity  of  this  living  barrier,  nor  would  it  explain  the  rapid 
disappearance  of  the  toxin  from  the  blood.  The  true  explanation 
is  certainly  that  the  toxin  combines  rapidly  with  the  cells  of  the 
body,  or  perhaps,  as  we  shall  see  later,  the  leucocytes,  and  is 
thereby  prevented  from  gaining  access  to  the  central  nervous 
system.  These  relatively  unimportant  cells  we  must  imagine  to 
have  the  same  nutritive  relations  to  the  molecules  of  toxin  as  have 
the  cells  of  the  heated  alligator ;  the  two  can  unite,  but  the  proto- 
plasm is  neither  injured  nor  nourished.  It  seems  probable  that  this 


IMMUNITY   TO   TOXINS  137 

affords  a  sufficient  explanation  of  the  differences  in  susceptibility 
of  the  "  susceptible  "  animals  which  figure  in  Knorr's  list,  and  that 
the  cells  of  the  central  nervous  system  in  the  warm-blooded  verte- 
brates differ  but  little  in  their  relation  to  tetanus  toxin.  Thus,  in 
the  case  of  the  horse  and  the  guinea-pig  little  toxin  is  bound  to  the 
body  cells,  and  practically  the  whole  amount  makes  its  way  to  the 
brain  wherever  the  injection  is  made.  In  the  rabbit  the  body  cells 
can  absorb  more,  so  that  if  only  a  small  dose  is  given  none  may 
reach  the  more  important  and  susceptible  organs.  The  fowl  marks 
the  other  end  of  the  scale ;  the  tissue  cells  must  have  an  enormous 
avidity  for  toxin,  and,  unless  absolutely  gigantic  doses  are  given, 
absorb  the  whole  of  it. 

The  objection  may  be  raised  that,  the  receptors  of  the  body  cells 
being  of  the  same  nature  as  antitoxin,  the  resulting  combination 
of  body-cell  receptor  and  toxin  should  undergo  dissociation,  the 
poison  being  gradually  passed  on  to  the  central  nervous  system, 
in  the  cells  of  which  dissociation  does  not  occur,  since  poisoning 
takes  place  ;  this  refers  to  the  case,  e.g.,  of  the  fowl.  It  is  true 
that  such  a  process  appears  to  occur  in  vitro.  If  tetanus  toxin  be 
mixed  with  emulsions  of  many  of  the  tissues,  the  two  enter  into  a 
loose  combination,  so  that  if  the  fragments  of  toxin-charged  tissues, 
after  thorough  washing  in  normal  saline  solution,  are  allowed  to 
soak  in  that  fluid,  toxin  is  gradually  liberated.  It  is  only  in  the 
case  of  the  central  nervous  system  that  stable  non-dissociable 
compounds  are  formed.  Several  interpretations  of  the  apparent 
anomaly  might  be  suggested.  It  is,  for  instance,  very  probable 
that  here,  as  in  so  many  other  phenomena  in  immunity,  the 
real  defensive  cell  is  the  leucocyte.  Thus,  Metchnikoff  found  that 
if  he  injected  tetanus  toxin  into  the  fowl,  and  then,  after  an  appro- 
priate interval,  excited  an  aseptic  exudate  composed  largely  of 
leucocytes,  the  fluid  thus  obtained  would  excite  tetanus  in  a  sus- 
ceptible animal.  Perhaps  the  chain  of  phenomena  is  as  follows  : 
The  toxin  first  unites  with  the  connective  and  other  tissue  cells, 
and  so  the  amount  that  the  leucocytes  have  to  deal  with  at  first  is 
greatly  diminished ;  dissociation  takes  place,  and  there  is  a  steady 
stream  of  toxin  from  the  tissues  to  the  blood.  This  is  dealt  with 
by  the  leucocytes,  which  thus  have  time  to  increase  in  numbers 
(and  even  in  the  insusceptible  fowl  the  result  of  the  injection  is  to 
cause  a  marked  leucocytosis),  and  perhaps  to  become  immune.  It 
is  useless  to  deny  that  these  phenomena  are  difficult  to  harmonize 
with  the  side-chain  theory  as  first  expounded,  or  that  Metchnikoff 


138  NATURAL   IMMUNITY   TO   TOXINS 

has  brought  forward  very  strong  (though  not  conclusive)  evidence 
in  support  of  his  theory  of  the  leucocytic  origin  of  antitoxin  ;  and, 
as  we  shall  see,  as  far  as  our  knowledge  goes,  we  are  led  to  believe 
that  all  antibodies  are  derived  from  the  spleen,  bone-marrow,  and 
other  organs  rich  in  leucocytes. 

The  study  of  the  affinity  of  bacterial  toxins  and  of  other  poisons 
to  various  tissues  and  organs  of  the  body  is  destined  to  play  a  part 
of  great  importance  in  our  ideas  of  pathology  and  pharmacology. 
It  has  been  especially  investigated  by  Ehrlich,  and  in  connection 
with  immunity  and  susceptibility  to  toxins  he  recognizes  four 
conditions  which  may  occur : 

1.  In  which  no   specific  receptors  occur  in  any  part  of   the 
animal.     This  is  the  first  form  of  natural  immunity  which  we 
have  discussed ;    the  animal  is  absolutely  immune,  and  cannot 
produce  antitoxin. 

2.  Receptors   are   present,  but  only  in  tissues  on  which   the 
poison  does  not  act,  or  in  tissues  of  little  importance.     Here  the 
animal  is  immune,  but  antibodies  may  be  produced.     This  is  the 
case  of  the  fowl  vis-a-vis  the  tetanus  toxin. 

3.  The   receptors   are   distributed   over   various   parts   of   the 
organism,  and  are  present  in  the  organs  which  are  sensitive  to  the 
action  of  the  poison.     Here  there  is  a  relative  immunity,  and  the 
degree  of  intoxication  depends  largely  on  the  region  into  which 
the   toxin   is   introduced.      These   animals   may   be   immunized, 
though  the  process  is  one  of  some  difficulty,  owing  to  the  sensitive- 
ness to  the  toxin  of  vital  organs,  and,  of  course,  antibodies  may 
be  produced. 

4.  The  receptors  are  present  only  in  vital  organs  which  are 
sensitive  to  the  poison.     Here  the  animal  is  very  susceptible,  and 
immunization  very  difficult,  involving  the  use  of  extremely  minute 
doses  or  of  toxoids.     The  action  of  tetanus  toxin  on  the  guinea- 
pig  and  horse  provides  examples. 


CHAPTER  VII 
BACTERIOLYSIS  AND  ALLIED  PHENOMENA 

UP  to  the  present  we  have  dealt  entirely  with  the  mechanism  by 
which  the  injurious  effects  of  the  infective  bacteria  are  nullified. 
It  is  obvious  that  this  is  only  one,  though  perhaps  the  most  im- 
portant, aspect  of  the  question.  We  have  now  to  study  how  the 
bacteria  themselves  are  removed. 

The  bactericidal  properties  of  the  blood  attracted  attention  very 
early  in  the  history  of  bacteriology,  and  long  before  the  beginnings 
of  the  science  Hunter  showed  that  blood  had  the  power  of  resisting 
decomposition  longer  than  other  animal  fluids.  It  was,  however, 
the  controversy  which  took  place  between  Metchnikoff  and  the 
humoralist  school  which  first  focussed  attention  on  this  question, 
and  led  to  the  discovery  of  the  alexins  by  Buchner,  Nuttall,  and 
others.  The  controversy  is  best  discussed  subsequently,  and  it  is 
sufficient  now  to  point  out  that  it  was  found  that  the  circulating 
blood  had  the  power  of  killing  certain  bacteria,  and  that  this 
property  was  even  more  marked  in  the  serum.  Thus,  according 
to  Lubarsch,  16,000  virulent  bacilli  will  kill  a  rabbit  if  injected 
intravenously — i.e.,  the  blood  has  not  the  power  of  killing  this 
number ;  yet  i  c.c.  of  fresh  serum  will  destroy  this  number  or 
more.  It  is  obvious  that  the  bactericidal  substance  or  substances 
occur  in  the  blood,  and  to  a  greater  extent  in  the  serum. 

The  properties  of  these  bactericidal  substances  or  alexins  were 
investigated,  and  it  was  found  that  they  were  fragile  bodies,  readily 
destroyed  by  a  moderate  temperature  (55°  C.),  and  that  they 
disappeared  spontaneously  if  the  serum  were  kept  for  a  few  days. 
They  were  destroyed  by  acids  and  alkalis,  and  what  was  most 
important  of  all  was  that  they  were  selective  in  their  action — i.e., 
those  from  a  certain  animal  might  be  potent  antiseptics  as  regards 
certain  bacteria  and  inert  towards  others,  whereas  the  serum  of 
another  species  might  have  quite  different  actions.  They  appeared 
to  be  formed  by  the  breaking  down  of  leucocytes  ;  hence  their 

139 


140 

appearance  in  the  blood  after  clotting  (when  fibrin  ferment  is  also 
liberated  from  these  cells),  and  their  absence  from  fluids  containing 
none,  such  as  the  aqueous  humour. 

It  was  obvious  that  this  was  a  foundation  for  a  theory  of 
immunity,  but  it  soon  became  apparent  that  it  did  not  explain  the 
whole  of  the  phenomena.  An  animal  might  be  immune  to  a 
certain  organism,  yet  its  serum  might  not  be  bactericidal  for  it,  or 
it  might  be  susceptible  and  yet  possess  suitable  alexins.  Thus, 
rabbits  are  very  susceptible  to  anthrax,  yet  have  serum  which 
kills  the  bacilli  in  large  numbers,  whilst  the  dog  is  much  less 
susceptible,  though  its  serum  is  very  slightly  bactericidal.  Such 
facts  prevented  the  alexic  theory  of  immunity  from  making  head- 
way until  the  discovery  of  Pfeiffer's  phenomenon. 

Pfeiffer  found  that  when  cholera  vibrios  were  injected  into  the 
peritoneal  cavity  of  a  highly  immunized  guinea-pig  they  were  not 
only  killed,  but  dissolved,  and  this  with  great  rapidity.  The 
vibrios  are  first  rendered  immotile,  and  then  lose  their  proper 
shape,  becoming  converted  into  spherical  masses  which  stain 
badly.  In  a  little  while  they  become  smaller,  and  disappear  alto- 
gether, no  trace  being  left. 

Pfeiffer  showed  that  the  phenomenon  could  also  be  produced 
by  injecting  into  the  peritoneum  of  a  normal  guinea-pig  a  mixture 
of  serum  from  an  immunized  animal  and  the  culture  of  vibrios. 
This  happened  when  an  old  specimen  of  serum  in  which  the 
alexins  had  disappeared  spontaneously  was  used,  or  a  fresh 
specimen  that  had  been  heated  to  60°  C.  Lastly,  he  found  that 
if  an  old  immune  serum  were  injected  into  the  peritoneal  cavity 
and  allowed  to  remain  for  a  while,  it  regained  its  bactericidal 
powers,  and  could  then  dissolve  the  vibrios  in  vitro  at  the  body 
temperature.  The  phenomenon  was  specific — i.e.,  if  serum  from 
an  animal  immunized  against  cholera  were  used  as  described 
above,  the  vibrios  of  cholera  were  dissolved,  but  no  others.  If 
typhoid  serum  were  used,  typhoid  bacilli  showed  degenerative 
changes,  though  they  were  not  completely  destroyed,  but  there 
was  no  effect  on  cholera  vibrios. 

The  explanation  which  suggested  itself  to  Pfeiffer  was  this  : 
There  is  in  the  serum  of  highly  immunized  animals  a  substance 
which  can  exist  either  in  an  active  or  inert  state.  In  the  blood- 
serum  or  peritoneal  fluid  whilst  in  the  body  it  occurs  as  an  active 
substance,  but  it  passes  into  the  inert  condition  when  kept  for  a 
few  days,  or  very  rapidly  when  heated  to  60°  C.,  and  this  inactive 


BACTERIOLYSIS   AND   ALLIED    PHENOMENA  141 

substance  can  be  rendered  active  again  by  contact  with  the  living 
endothelial  cells  of  the  peritoneum. 

The  true  explanation  was  given  by  Bordet,  who  showed  that 
two  substances  are  necessary  for  the  phenomenon.  The  one  is  the 
inert  thermostable  substance  which  occurs  in  the  serum  of  highly 
immunized  animals,  but  not  in  normal  animals,  and  which  does 
not  disappear  on  keeping.  The  second  is  a  thermolabile  sub- 
stance which  occurs  in  fresh  serum,  whether  from  a  normal  or 
from  an  immunized  animal.  This  he  showed  by  demonstrating 
that,  whereas  neither  heated  nor  stale  immune  serum  alone,  nor 
fresh  serum  alone,  had  the  power  of  leading  to  the  production  of 
Pfeiffer's  reaction  in  vitro,  this  was  caused  when  the  two  were 
used  in  combination.  When  a  drop  of  fresh  serum  from  a  normal 
animal,  some  heated  immune  serum,  and  the  cholera  vibrios  are 
mixed  together  and  incubated,  the  whole  train  of  phenomena  was 
repeated. 

The  thermolabile  substance  taking  part  in  the  process  was 
soon  identified  as  the  alexin  of  the  earlier  investigators,  and  the 
new  substance  was  called  by  Bordet  substance  sensibilatrice.  His 
theory  was  that  alexin  alone  does  not  unite  with  the  bacteria, 
unless  these  have  been  previously  sensitized  by  the  action  of  the 
substance  in  the  immune  serum.  It  was  shown  that  the  substance 
sensibilatrice  has  the  power  of  uniting  with  the  bacteria  as  follows : 
A  culture  exposed  to  the  action  of  heated  immune  serum  and 
washed  by  repeated  centrifugalizations  with  normal  saline  solution 
is  apparently  unaltered,  but  the  bacteria  are  dissolved  by  the 
action  of  normal  serum,  which  has  no  action  on  unsensitized 
bacteria.  Bordet  supposed,  therefore,  that  the  immune  serum 
alters  the  constitution  of  the  bacteria  in  some  way,  so  that  the 
alexin  can  combine  with  them  subsequently.  He  compared  the 
process  to  the  opening  of  a  lock,  which  can  only  be  effected  by 
means  of  two  keys,  of  which  one  (the  sensibilatrice)  must  be 
turned  before  the  other  (alexin)  can  be  introduced. 

Bordet  found  that  the  essential  facts  could  be  reproduced 
exactly  if  red  blood-corpuscles  were  substituted  for  bacteria.  It 
was  previously  known  that  the  serum  of  some  animals  possesses 
the  power  of  liberating  the  haemoglobin  (haemolysis)  from  the  red 
corpuscles  of  other  species,  just  as  the  serum  of  some  animals 
can  destroy  certain  bacteria.  Bordet  showed  that  the  guinea-pig 
serum  has  normally  no  action  on  the  red  corpuscles  of  the  rabbit, 
but  that  it  becomes  haemolytic  to  the  latter  after  a  few  injections 


142  HAEMOLYSIS — BORDET'S  DISCOVERIES 

of  rabbit's  blood.  Just  as  an  animal  which  was  formerly  destitute 
of  that  power  becomes  able  to  dissolve  the  cholera  vibrio  after 
a  few  injections  of  that  organism,  so  a  guinea-pig,  the  serum  of 
which  had  formerly  no  action  on  the  rabbit's  corpuscles,  acquires 
the  power  of  dissolving  them  as  the  result  of  an  injection  or  two 
of  rabbit's  blood ;  hence,  following  the  analogy  with  the  cholera 
vibrio,  the  guinea-pig  is  said  to  be  "  immunized  "  to  the  rabbit's 
corpuscles,  though  there  is  here  no  question  of  the  avoidance  of 
any  deleterious  action.  Further — and  hence  the  importance  of 
these  discoveries  in  the  theory  of  immunity — Bordet  showed  that 
the  laws  which  govern  bacteriolysis  by  means  of  immune  sera 
were  apparently  identical  with  those  governing  Pfeiffer's  pheno- 
mena. The  fresh  immune  serum  is  haemolytic ;  it  loses  its  power  on 
heating  to  55°  C.,  and  it  regains  it  on  the  addition  of  fresh  normal 
serum.  Further,  the  action  is,  to  some  extent  at  least,  a  specific 
one.  An  animal  (A)  injected  with  corpuscles  of  another  species 
(B)  will  dissolve  the  corpuscles  of  that  species,  and  may  also  have 
some  action  on  those  of  animals  closely  allied  zoologically.  It  is 
evident  that  bacteriolysis  and  haemolysis  are  closely  akin,  and  as 
researches  on  the  latter  phenomena  are  infinitely  more  easy  than 
on  the  former,  much  work  intended  to  elucidate  the  mechanism 
of  immunity  has  been  carried  out  on  red  corpuscles.  This  is  to 
some  extent  regrettable,  since  the  processes,  though  similar,  are 
not  identical,  and  it  is  unsafe  to  argue  from  one  to  the  other 
without  experimental  verification. 

It  was  this  analogy  between  bacteriolysis  and  haemolysis  that 
led  Ehrlich  to  an  investigation  of  the  latter  phenomenon,  and 
his  researches  led  to  a  flood  of  new  light  being  thrown  upon  the 
subject.  Ehrlich  introduced  fresh  names  for  the  substances  which 
Bordet  had  shown  to  be  necessary  for  the  phenomenon,  and  it 
will  now  be  convenient  to  give  a  list  of  the  various  terms  which 
the  theories  or  caprices  of  various  writers  have  applied  to 
each. 

The  thermostable  substance  has  been  called : 

Substance  sensibilatrice,  or  simply  sensibilatrice. 
Immune  body.  Philocytase. 

Amboceptor.  Immunisin. 

Fixator.  Desmon. 

Intermediary  body.  Copula. 

Interbody.  Preparator. 


BACTERIOLYSIS   AND   ALLIED    PHENOMENA  143 

Whilst  the  thermolabile  substance  is  spoken  of  as : 

Alexin.  Complement. 

Addiment.  Cytase. 

We  shall  speak  of  the  substances  as  amboceptor  or  immune 
body  and  as  alexin  or  complement  respectively.    Objection  may  be 


FIG.  24. — THE   CONSTITUENTS  OF  FRESH    IMMUNE  SERUM,    ON    EHRLICH'S 

THEORY. 

a  =  complement,  b  =  amboceptor,  d  being  its  complementophile,  and  c  its  cyto- 
phile  groups.  Below,  a  red  blood-corpuscle,  showing  its  receptors. 

This  and  the  following  figures  are  modified  from  Ehrlich,  and  are  intended  to 
illustrate  his  theories  of  haemolysis.  The  black  figures  represent  normal 
substances  (corpuscles,  complement),  the  white  ones  antibodies.  It  need 
hardly  be  said  that  they  are  absolutely  diagrammatic. 


FIG.  25.  —THE  IMMUNE  SERUM  SHOWN  IN  FIG.  24,  AFTER  HEATING. 
The  complement  is  destroyed,  and  amboceptor  only  remains. 


FIG.  26.  —  NORMAL  SERUM  CONTAINING  COMPLEMENT,  BUT  NO  AMBOCEPTOR. 

taken  to  Ehrlich's  terms  "amboceptor"  and  "complement,"  since, 
as  we  shall  show,  they  imply  a  theory,  but  they  are  too  firmly 
rooted  to  be  displaced. 

Ehrlich  applied  his  side-chain  theory  to  the  study  of  the 
phenomena,  and  argued  that  amboceptor  must  be  an  antibody 
to  the  receptors  of  the  red  blood-corpuscle.  If  this  is  the  case, 
it  ought  to  unite  with  the  corpuscles,  and  Ehrlich  showed  in  the 


144 


HAEMOLYSIS — EHRLICH'S  RESEARCHES 


following  way  that  such  actually  occurred  :  He  worked  with  the 
serum  of  a  goat  which  had  been  injected  with  and  was  haemolytic  for 
the  red  blood-corpuscles  of  a  sheep.  He  heated  this  immune  serum 
to  56°  C.  to  destroy  complement,  and  added  some  sheep's  cor- 
puscles ;  the  mixture  was  then  centrifugalized,  the  supernatant  fluid 
then  pipetted  off,  and  replaced  by  normal  saline  solution.  The  red 
corpuscles  were  to  all  appearance  unaltered,  but  it  was  now  found 


FIG.  27. — HEATED   IMMUNE    SERUM    ADDED    TO    RED   BLOOD  -  CORPUSCLES, 

WHICH  ARE  APPARENTLY  UNALTERED,   BUT  ARE  IN  REALITY  "  SENSITIZED." 


FIG.  28. — EFFECT  OF  ADDITION  OF  FRESH  NORMAL  SERUM  TO  SENSITIZED 

CORPUSCLES. 

Complement  is  now  linked  up  to  the  corpuscles,  and  haemolysis  takes  place 
when  they  are  incubated  at  37°  C. 

that  if  a  small  amount  of  normal  goat  serum  were  added  and  the 
mixture  incubated,  haemolysis  occurred.  It  was  evident,  therefore, 
that  the  corpuscles  underwent  some  change  in  virtue  of  their  stay 
in  the  heated  immune  serum,  though  no  alteration  was  obvious. 

In  a  further  experiment  he  showed  that  the  change  consisted  in 
the  abstraction  of  the  amboceptor  from  the  fluid.  This  appeared 
from  the  fact  that  if  the  supernatant  fluid  from  the  last  experi- 
ment were  pipetted  off,  fresh  normal  goat's  serum  added  (to  supply 
complement),  and  the  mixture  tested  with  sheep's  corpuscles,  no 


BACTERIOLYSIS   AND   ALLIED    PHENOMENA  145 

haemolysis  occurred.     Evidently  it  had  been  removed  by  the  first 
addition  of  corpuscles. 

Red  corpuscles,  therefore,  have  a  combining  affinity  for  ambo- 
ceptor,  and  in  further  experiments  Ehrlich  showed  that  this 
combination  takes  place  at  ordinary  temperatures  or  at  low  ones, 
down  to  o°  C.  Expressed  in  the  language  of  the  side-chain  theory, 
amboceptor  has  a  haptophore  group  with  a  combining  affinity 
for  the  receptors  of  the  corpuscle,  bacterium,  etc.,  with  which  it 


'*'** 

*       t  ~ 


FIG.  29. — NORMAL  GOAT  SERUM  (COMPLEMENT),  PLUS  SHEEP'S  CORPUSCLES. 

No  combination.  This  is  shown  as  follows  :  The  mixture  is  centrifugalized, 
and  to  the  corpuscles  heated  immune  serum  (amboceptor)  was  added 
(Fig.  30),  whilst  to  the  supernatant  fluid  corpuscles  and  heated  serum 
were  added  (Fig.  31). 


FIG.   30. — THE   CORPUSCLES   FROM   THE    PREVIOUS   EXPERIMENT   INCUBATED 

WITH  HEATED  IMMUNE  SERUM. 

No  solution,  showing  that  no  complement  had  been  abstracted  in  combination 

with  them. 


unites.  We  shall  see  reasons  for  believing  that  it  may  have  a 
second  haptophore  group,  and  shall  distinguish  this  first  by  the 
name  of  cytophile  haptophore. 

Ehrlich  now  investigated  the  behaviour  of  the  second  substance 
— the  complement — with  the  red  corpuscles  by  a  precisely  similar 
method,  and  found  that  the  two  had  no  power  of  entering  into 
combination.  Thus,  normal  goat's  blood  (containing  complement) 
was  added  to  sheep's  corpuscles,  and  the  mixture  centrifugalized. 
To  the  corpuscles  heated  immune  serum  (amboceptor)  was  added, 
but  there  was  no  haemolysis.  Again,  to  the  supernatant  fluid 
sheep's  corpuscles  and  heated  serum  were  added,  and  haemolysis 

10 


146 


HAEMOLYSIS — EHRLICH'S  RESEARCHES 


i 


occurred  ;  complement  had  evidently  not  been  withdrawn  from  the 
fluid.  Complement,  therefore,  will  not  unite  with  red  corpuscles 
direct.  It  has  no  haptophore  group  with  an  affinity  for  the 
receptors  of  the  latter. 

This  led  Ehrlich  to  the  theory  that  the  complement  united  with 
the  red  corpuscle  only  indirectly  by  means  of  the  amboceptor. 


FIG.  31. — THE  SUPERNATANT  FLUID  FROM   FIG.   29,  TESTED  WITH  HEATED 
IMMUNE  SERUM  AND  SHEEP'S  CORPUSCLES. 

Solution  takes  place,  showing  that  the  complement  had  not  been  removed. 


FIG.    32. — FRESH    IMMUNE    SERUM   (OR   A    MIXTURE   OF    HEATED    IMMUNE 
SERUM  AND  FRESH  NORMAL  SERUM)  ADDED  TO  CORPUSCLES  AT  o°  C. 

Amboceptor  unites  therewith,  complement  does  not. 

He  pictured  the  latter  as  having  two  haptophore  groups :  a  cyto- 
phile,  which  we  have  already  mentioned,  and  a  complementophile, 
with  which  the  complement  could  unite  after  the  former  had 
seized  on  a  receptor  of  the  red  corpuscle.  The  process  of  haemo- 
lysis was  supposed  to  take  place  as  follows  :  In  fresh  immune 
serum  or  in  a  mixture  of  heated  immune  serum  and  fresh  normal 
serum  (i.e.,  of  amboceptor  and  complement)  the  two  substances 
occur  independently  of  one  another.  When  the  mixture  is  kept 
in  the  cold,  and  red  corpuscles  are  added,  the  cytophile  groups  of 
the  amboceptor  molecules  attach  themselves  to  the  receptors  of 


BACTERIOLYSIS    AND    ALLIED    PHENOMENA  147 

the  red  corpuscles,  but  the  molecules  of  the  complement  still 
remain  free,  and  do  not  attach  themselves  either  directly  or  in- 
directly to  the  corpuscles.  Nevertheless,  there  is  a  change  in  the 
combining  affinities  of  the  complementophile  haptophore.1  This 
is  shown  by  the  fact  that  if  the  mixture  be  raised  to  the  body- 
heat,  the  molecules  of  complement  attach  themselves  to  these 
haptophores,  exert  a  digestive  or  haemolytic  action  through  the 
amboceptor  on  the  red  corpuscles,  and  haemolysis  occurs.  Hence, 
on  Ehrlich's  theory,  the  complement  does  not  attack  the  cell  or 
corpuscle  direct  (as  Bordet  holds,  and  as  is  implied  in  the  term 
substance  sensibilatrice  for  amboceptor),  but  it  unites  itself  to 
one  part  of  the  molecule  of  amboceptor  after  another  part  of  the 
latter  has  combined  with  the  corpuscle.  We  will  defer  the 
discussion  of  these  theories  for  the  present. 

For  the  sake  of  simplicity,  we  have  assumed  that  complement 
will  not  unite  with  amboceptor  until  the  latter  has  combined  with 
its  antigen.  Ehrlich,  however,  assumes  (on  no  very  clear  evidence) 
that  the  two  substances  may  enter  into  a  loose  and  easily  dis- 
sociated chemical  combination.  This  is  hastened  by  heat  and 
retarded  by  cold.  The  union  between  corpuscle  and  amboceptor 
is  a  firm  one,  which  takes  place  at  low  temperatures,  and  which 
does  not  tend  to  dissociate,  and  after  the  combination  amboceptor 
has  a  stronger  affinity  for  complement,  though  even  then  a  firm 
union  only  takes  place  at  30°  C.  or  over. 

Ehrlich  explains  the  method  of  formation  of  amboceptor  on  the 
side-chain  theory  in  this  wise :  He  points  out  that  for  the 
absorption  of  substances  of  small  molecule  by  the  cell  protoplasm 
the  simple  receptors,  which,  when  cast  off,  constitute  antitoxin, 
may  suffice ;  but  he  says  that  when  a  giant  proteid  molecule  (e.g., 
of  albumin)  is  anchored,  it  requires  the  action  of  a  digestive 
ferment  before  it  can  be  brought  into  a  condition  to  be  of  use 
in  the  nourishment  of  the  living  cell.  Hence  it  must  be  first 
broken  down  by  means  of  a  proteolytic  enzyme,  and  it  is  of  this 
nature  that  he  imagines  the  complement  to  be.  He  holds,  there- 
fore, that  the  receptors  or  side-chains  which  are  adapted  to  seize 
molecules  of  coagulable  proteid  possess  two  haptophore  groups, 
the  one  to  seize  the  nutrient  material,  and  the  other  to  seize  a 
molecule  of  digestive  enzyme  from  the  surrounding  blood  or 
lymph.  When  the  formation  of  such  receptors  is  stimulated  to 

1  Ehrlich  does  not  make  a  definite  statement  as  to  this  increase  of  affinity, 
but  it  seems  a  necessary  deduction  from  the  facts  as  he  interprets  them. 

10 — 2 


148  HAEMOLYSIS — EHRLICH'S  RESEARCHES 

excess,  and  they  break  loose,  they  retain  both  their  haptophore 
groups  and  constitute  amboceptor.  The  stimulation  and  over- 
production is,  of  course,  produced  by  the  molecules  of  the  substance 
injected  (the  specific  antigens),  be  they  red  blood-corpuscles, 
bacteria,  or,  as  we  shall  see,  a  whole  host  of  other  cells. 

As  a  rough  illustration  of  the  nature  of  these  seizing  arms 
Ehrlich  compares  them  with  the  tentacles  of  Drosera,  which 
seize  the  nutrient  material  and  then  secrete  a  digestive  fluid. 
The  analogy  is  not  exact,  since  the  receptors  do  not  form  the 
digestive  enzyme,  but  simply  select  it  from  without. 

Ehrlich  compares  the  action  of  the  haemolysins  with  that  of 
the  toxins,  and  points  out  that  in  each  case  there  is  a  haptophore 
group  adapted  to  seize  on  the  red  corpuscle  to  be  dissolved  or  the 
cell  to  be  poisoned,  and  an  actively  functional  group  with  functions 
resembling  those  of  an  enzyme.  He  says  that  the  haemolysins 
are  practically  toxins  in  two  parts,  and  that  a  combination  of  these 
parts  is  necessary  before  any  action  can  be  effected. 

These  discoveries  and  theories  of  Ehrlich  led  to  a  series  of 
further  researches,  all  of  which  were  directly  suggested  by  them, 
and  the  results  of  which  appear  to  corroborate  them  to  the  full. 
Whatever  the  ultimate  fate  of  the  side-chain  theory,  there  can  be 
no  doubt  that  it  has  lead  to  an  enormous  increase  of  our  knowledge 
on  a  most  intricate  subject.  These  researches  have  in  many 
cases  but  little  direct  bearing  at  present  on  the  subject  of 
immunity,  but  they  are  far  too  important  to  be  passed  over 
without  mention.  We  shall,  therefore,  summarize  them  as  briefly 
as  is  possible  when  dealing  with  so  complicated  a  subject.  We 
will  deal  first  with  the  explanation  of  the  haemolytic  action  which 
the  serum  of  some  normal  animals  has  on  the  corpuscles  of  other 
species ;  for  instance,  normal  goat  serum  will  dissolve  the  red 
corpuscles  of  the  guinea-pig.  This  was  shown  conclusively  to 
depend  upon  the  same  mechanism  of  amboceptor  and  complement 
as  in  the  artificial  immune  sera.  The  proof  was  as  follows : 
Goat  serum  was  mixed  with  guinea-pig's  corpuscles,  and  the 
mixture  kept  at  o°  C.,  and  then  centrifugal ized.  The  theory  would 
lead  us  to  believe  that  the  amboceptor  had  combined  with  the 
corpuscles,  but  that  the  complement  had  remained  free.  If  this 
were  so,  the  supernatant  fluid  would  be  found  to  have  lost 
amboceptor.  To  test  this  a  further  addition  of  corpuscles  was 
made,  and  the  solvent  power  of  the  serum  for  these  was  found 
to  be  lowered.  Evidently,  then,  sera  which  are  naturally  haemo- 


BACTERIOLYSIS    AND    ALLIED    PHENOMENA  149 

lytic  to  certain  red  corpuscles  are  so  in  virtue  of  containing 
amboceptors  with  cytophile  haptophore  groups  which  fit  the 
receptors  of  these  cells  and  complements  which  can  dissolve 
them.  He  was  also  able  to  show,  by  an  ingenious  experiment, 
that  when  a  normal  serum  can  haemolyze  the  corpuscles  from 
different  species  of  animals,  it  does  so  by  the  action  of  different 
amboceptors,  and  in  some  cases  of  different  complements  also. 
(,We  must  imagine,  therefore,  that  normal  serum  contains  numerous 
antibodies  of  the  amboceptor  type,  and  adapted  to  dissolve 
numerous  foreign  substances  when  they  gain  access  to  the  blood ; 
and  it  seems  reasonable  to  believe  that  these  normal  antibodies 
and  those  which  are  formed  as  a  result  of  the  presence  of  the 
foreign  substances  play  a  part  of  the  greatest  importance  in 
immunity. \ 

The  question  then  arose,  Do  animals  possess  or  form  haemolysins 
adapted  to  the  solution  of  their  own  corpuscles — in  other  words, 
an  autohtsmolysin  ?  For  instance,  if  a  person  sustains  a  large 
internal  haemorrhage,  and  the  blood  is  absorbed,  is  he  thereby 
stimulated  to  the  production  of  a  substance  which  dissolves  his 
own  red  corpuscles  ?  Ehrlich  pointed  out  that  such  a  phenomenon 
is  unknown,  and  set  out  to  investigate  the  reason  of  its  non- 
occurrence.  He  selected  goats  as  his  experimental  animals,  and 
injected  into  them  large  amounts  of  mixed  blood-corpuscles  from 
other  goats.  In  a  week's  time  the  serum  was  found  to  be  power- 
fully haemolytic,  but  only  towards  the  corpuscles  of  other  goats, 
never  towards  its  own;  thus  the  serum  of  the  first  animal 
experimented  on  was  found  to  dissolve  the  corpuscles  of  goats  i, 
2,  4,  5,  6,  and  9  easily ;  those  of  3  and  8  less  powerfully ;  and 
those  of  7  and  of  itself  not  at  all.  A  second  animal  was  treated 
just  like  the  first,  and  it  also  developed  a  haemolysin,  but  this 
did  not  act  on  its  own  corpuscles,  nor  on  those  of  other  goats 
in  the  same  way  as  that  of  the  first ;  evidently  a  different  series 
of  amboceptors  had  been  formed.  Ehrlich  speaks  of  a  haemolysin 
formed  by  the  injection  of  the  corpuscles  of  a  different  species 
as  heterolysin,  that  which  acts  on  the  blood  of  the  same  species 
as  an  isolysin ;  one  which  would  act  on  the  blood-corpuscles  of 
the  same  animal  would  be  called  an  autolysin,  but  this  is  never 
formed. 

In  attempting  to  discover  the  reason  for  this  non-formation 
Ehrlich  first  proved  that  the  amboceptor  of  the  isolysin  was 
anchored  by  the  corpuscles  which  it  dissolved,  but  not  by  those 


150  HAEMOLYSIS — ISOLYSIN 

of  the  goat  from  which  it  was  derived.  An  obvious  explanation 
would  be  that  .the  latter  did  not  possess  any  receptors  which  it 
would  fit.  Another  suggestion  might  be  that  it  had  such  receptors, 
but  that  they  were  already  fully  occupied  by  amboceptors  occur- 
ring in  the  serum  ;  but  in  this  case  they  would  attract  complement, 
and  solution  would  ensue.  By  a  method1  which  will  not  be 
described  here,  since  it  would  involve  anticipation  of  facts  not 
yet  described,  Ehrlich  was  able  to  prove  the  former  view  the 
correct  one.  The  immunity,  therefore,  of  the  corpuscles  of 
an  animal  to  its  own  isolysin  is  due  to  a  complete  absence 
of  suitable  receptors  in  its  corpuscles,  and,  we  may  add,  in  the 
entire  organism. 

He  deduced,  therefore,  that  each  blood-corpuscle  possesses 
numerous  side-chains  with  haptophore  groups,  each  of  which, 
when  injected  into  a  living  animal,  is  able  to  combine  with  a 
suitable  receptor.  If  we  take  a  particular  variety  of  haptophore, 
which  we  will  call  a,  we  can  see  that  there  are  two  possibilities 
after  the  injection ;  it  may  find  no  receptors  a,  in  which  case 
there  will  be  no  antibody  formed,  or  it  may  find  such  receptors. 
In  the  latter  case  there  are  also  two  possibilities :  there  may  be 
receptors  a  only,  or  there  may  be  receptors  a  and  haptophore  side- 
chains  a. 

If  there  are  only  receptors  a  and  no  side-chains  a,  the  injection 
of  corpuscles  with  side-chains  a,  will  lead  to  an  overproduction  of 
receptors  a,  and  amboceptor  will  be  produced.  This  will  act  as  a 
hsemolysin  to  the  corpuscles  injected,  but  not  to  those  of  the 
animal  itself,  since  they  do  not  contain  side-chains  to  which  it  can 
attach  itself ;  it  will  be  an  isolysin,  not  an  autolysin. 

In  the  second  possible  case  Ehrlich  points  out  that  the  condi- 
tions for  the  production  of  an  autolysin  do  occur,  and  such  a 
substance  might  be  produced  and  might  do  serious  harm  to  the 
animal.  But  it  would  be  produced  at  first  only  in  small  amounts, 
and  the  result  might  be  that  it  would  combine  with  receptor  a, 
which  might  then  be  stimulated  and  cast  off  and  would  form  anti- 
autolysin. 

1  He  first  showed  that  the  injection  of  an  amboceptor  from  one  animal  into 
another  of  a  different  species  would  cause  the  production  of  an  anti-ambo- 
ceptor,  and  then  showed  that  the  injection  of  a  serum  containing  isolysin 
into  a  goat  whose  corpuscles  it  dissolved  produced  anti-isolysin.  These 
corpuscles  contained  receptors,  since  they  fixed  the  isolysin.  In  the  goat 
from  which  the  isolysin  came  there  was,  of  course,  no  production  of  anti- 
isolysin.  Hence,  he  argued,  it  had  no  suitable  receptors  in  its  entire  body. 


BACTERIOLYSIS    AND    ALLIED    PHENOMENA  151 

There  are,  therefore,  three  possibilities  :  no  formation  of  haerno- 
lysin,  the  formation  of  an  isolysin,  and  the  formation  of  an  anti- 
autolysin.  In  other  words,  if  autolysins  were  ever  developed  they 
would  be  followed  immediately  by  the  production  of  their  specific 
antibody. 

But  Ehrlich  assumes — and  our  previous  account  of  the  iso- 
agglutinins  leads  us  to  be  ready  to  accept  his  assumption 
unhesitatingly — that  each  corpuscle  in  every  animal  has  numerous 
side-chains,  «,  (3,  7,  etc.  If  such  a  corpuscle  is  injected  into 
an  animal  of  the  same  species,  a  may  lead  to  no  result,  finding  no 
receptors  ;  ft  may  lead  to  the  production  of  an  isolysin,  and  7  to 
that  of  an  anti-autolysin.  The  existence  of  the  latter  substance 
could  not,  of  course,  be  proved,  since  it  was  never  possible  to 
prepare  autolysin. 

Hence  what  Ehrlich  calls  the  "  pluralistic  conception  of  the 
cellular  immunity  reaction."  There  are,  he  says,  in  each  bac- 
terial cell  numerous  side-chains,  to  each  of  which  an  antibody 
is  theoretically  possible,  and  an  ideal  curative  serum  would 
contain  all  these  antibodies.  But  in  some  cases,  perhaps  in  most, 
only  a  few  are  found,  and  others  cannot  be  developed ;  for 
instance,  in  some  animals  it  is  impossible  to  produce  anti -enzymes 
by  the  injection  of  enzymes.  He  explains  this  in  two  ways. 
The  specific  receptors  may  be  of  peculiar  constitution,  so  that  they 
cannot  be  cast  off  from  the  cell  in  the  process  of  immunization : 
these  he  calls  sessile  receptors.  Secondly,  it  is  conceivable  that 
the  side-chains  in  question  are  normally  produced  by  the  animal 
cells,  and  that  no  antibody  is  produced  to  them,  or,  at  any  rate,  that 
none  accumulates. 

But  when  either  or  both  of  the  conditions  arise,  we  may  be  able 
to  get  the  antibodies  from  a  second  animal  which  we  are  unable 
to  do  from  the  first.  Thus,  if  we  imagine  that  the  typhoid  bacillus 
has  twenty  different  sorts  of  side-chains,  we  may  be  able  to  get 
antibodies  to  some  of  them  from  the  dog,  to  others  from  the  rabbit, 
etc.  Hence  he  suggests  as  an  important  principle  in  the  forma- 
tion of  curative  sera  to  use  many  animal  species,  mixing  the  sera 
of  those  which  produce  the  antibodies  required  for  use.  (This,  it 
must  be  pointed  out,  is  not  what  is  meant  by  a  polyvalent  serum, 
which  is  a  serum  formed  by  the  injection  of  many  strains  of  the 
same  organism.) 

Leaving  this  subject  for  the  present,  we  will  pass  on  to  Ehrlich's 
researches  into  the  nature  of  complements.  Buchner,  in  his 


152  HAEMOLYSIS — COMPLEMENT 

studies  on  the  alexins,  assumed  that  the  serum  of  each  animal 
contained  one  alexin,  though  the  alexins  of  different  animal 
species  were  different.  This  view  was  also  held  by  Bordet  and 
others,  and  constitutes  the  "  Unitarian  "  view  of  alexin  or  com- 
plement. Metchnikoff  holds  that  there  are  two  alexins,  or,  as  he 
calls  them,  cytases — macrocytase,  which  acts  specially  on  red 
blood-corpuscles,  cells,  etc.,  and  i /jSaeJ  by  the  large  mononuclear 
cells ;  and  microcytase,  which  acts  on  bacteria,  and  is  formed  by  the 
polynuclear  leucocytes. 

As  against  these  views,  Ehrlich  advances  the  theory  of  the 
multiplicity  of  the  complements,  the  proof  of  which  appears  entirely 
satisfactory.  According  to  him  there  are  numerous  complements 
in  the  serum  of  every  animal,  and  these  differ  from  one  another  in 
their  haptophore  groups,  so  that  one  can  reactivate  one  ambo- 


*  3'  # 


FIG.  33.— SHOWING  SOME  OF  THE  CONSTITUENTS  WHICH  CAN  BE  DEMON- 
STRATED IN  THE  SERUM  OF  A  GOAT  WHICH  HAS  BEEN  INJECTED  WITH 
SHEEP'S  CORPUSCLES. 

a,  b,  and  c  are  the  different  complements,  a' ,  b' ,  and  c'  the  amboceptors  which 
unite  them  to  the  corpuscles  of  the  sheep  (A),  rabbit  (B),  and  guinea  pig  (C). 

ceptor,  another  another.  They  differ  in  some  cases  in  other 
respects.  The  first  example  which  he  was  able  to  adduce  in 
which  there  were  at  least  two  complements  in  a  sample  of  serum 
was  that  of  a  goat  which  had  been  injected  with  sheep's  corpuscles, 
and  which  dissolved  those  of  the  sheep,  guinea-pig,  and  rabbit. 
But  when  this  serum  was  heated  to  56°  C.  for  three-quarters  of  an 
hour,  it  was  found  to  have  no  action  on  the  corpuscles  of  the  latter 
animals,  whilst  that  of  the  former  was  unchanged.  Evidently 
then,  the  complement  which  activated  the  amboceptor  combining 
with  the  sheep's  corpuscles  was  thermostable  ;  that  acting  on  the 
others,  thermolabile.  This  was  the  first  known  example  of  a 
thermostable  complement.  In  a  later  experiment  he  was  able  to 
prove  that  the  complements  taking  part  in  the  haemolysis  of 
rabbit's  corpuscles  and  that  taking  part  in  the  solution  of  guinea- 
pigs  were  different :  the  latter  passes  through  a  Pukall's  filter, 
the  former  does  not. 


BACTERIOLYSIS    AND    ALLIED    PHENOMENA  153 

These  methods  of  heating,  filtration,  etc.,  of  course  only  separate 
the  complements  in  certain  lucky  cases,  and  the  more  general 
method  involves  the  removal  of  some  of  these  bodies  by  means  of 
sensitized  red  corpuscles.  Thus  fresh  goat  serum  will  reactivate 
heated  normal  goat  serum  in  its  action  on  rabbits  and  on  guinea- 
pig's  red  corpuscles,  the  amboceptors  for  which  are  contained  in 
untreated  animals.  It  will  also  reactivate  the  heated  serum 
of  goats  injected  with  rabbit's  corpuscles,  ox  corpuscles,  or  dog's 
corpuscles.  Ehrlich  and  Sachs  found  that  if  they  added  this  fresh 
serum  to  rabbit's  corpuscles,  the  complements  which  took  part  in 
the  two  latter  reactions  were  absorbed,  whilst  the  others  were 
unaltered.  Evidently,  therefore,  two  complements  at  least  are 
present  in  goat  serum.  When  the  serum  was  allowed  to  act 
on  guinea-pig's  corpuscles,  the  complements  which  reactivated 
the  normal  amboceptors  for  rabbit's  and  guinea-pig's  corpuscles 
were  removed,  also  that  which  activated  the  artificial  ox  ambo- 
ceptor.  By  an  elaboration  of  these  methods  Ehrlich  and  Sachs 
were  able  to  demonstrate  that  these  five  hsemolytic  actions  depend 
upon  five  different  complements,  each  perfectly  distinguishable 
the  one  from  the  other.  The  subject  will  not  be  pursued  farther, 
though  there  is  an  abundance  of  evidence  pointing  to  the  same 
end — that  the  serum  of  every  animal  contains  a  very  large  number 
of  complements,  some  of  which  take  part  in  one  reaction,  some  in 
another.  As  a  rule,  complements  which  take  part  in  haemolysis 
have  no  action  on  bacteria,  or  but  little,  and  this  is  all  the  truth  in 
MetchnikofT's  theory  of  macro-  and  microcytase.1 

Ehrlich  lays  it  down  as  a  law  that  amboceptors  from  a  certain 
species  of  animal  are  best  complemented  by  complements  from 
this  species,  and  this  is  true  in  general  ;  Muir  has  adduced  some 
exceptions. 

The  main  experimental  basis  for  the  Unitarian  theory  was 
furnished  by  Bordet  and  Gengou.  They  found  that  if  they  added 
"sensitized"  bacteria — i.e.,  bacteria  which  had  been  placed  in 
heated  immune  serum — in  fresh  serum,  all  the  complements  were 
removed,  and  the  fluid  would  no  longer  dissolve  sensitized  red 
corpuscles,  so  that  evidently  the  haemolytic  complement  had  been 
removed.  Again,  Bordet  showed  that  if  he  added  fresh  serum  to 
sensitized  red  corpuscles  and  allowed  solution  to  take  place,  all 
complements,  bacteriolytic  as  well  as  haemolytic,  were  removed. 
These  facts  are  not  disputed,  and  we  shall  have  occasion  to  refer 

1  See  p.  286. 


154  HAEMOLYSIS — COMPLEMENT 

to  them  again,  as  they  are  of  great  importance.  Their  interpreta- 
tion is,  however,  still  uncertain,  and  they  cannot  outweigh  the 
very  precise  demonstrations  of  numerous  complements  by  Ehrlich 
and  his  school.  According  to  them  the  receptor,  which,  when 
broken  off,  constitutes  amboceptor,  may  have  more  than  one  com- 
plementophile  group,  each  adapted  to  seize  and  utilize  different 
complements.  We  must  suppose  that  it  can  discharge  its  function 
if  it  is  supplied  with  one  of  these  complements  ;  this  is  called  the 
dominant.  It  may  also  seize  other  complements,  so  that  all  its 
affinities  are  supplied,  and  this  is  what  takes  place  in  Bordet's 


FIG.  34. — PLURICEPTORS   ATTACHED   TO    A   CORPUSCLE,  AND   SHOWING    THE 
DOMINANT  (a)  AND  NON-DOMINANT  (b)  COMPLEMENTS. 


FIG.  35.—  SOME  OF  THE  CONSTITUENTS  OF  NORMAL  GOAT  SERUM. 

a  —  Complement  which  dissolves  sensitized  sheep-corpuscles;  b  =  that  which 
dissolves  rabbit's  corpuscles  ;  c  =  the  normally  occurring  amboceptor  for 
rabbit's  corpuscles.  The  next  two  diagrams  shows  how  this  is  shown  to 
be  a  biceptor. 

and  Gengou's  phenomenon  ;  these  complements,  which  are  not 
essential  to  the  lytic  action,  are  called  non-dominant  or  subordinate 
complements. 

Ehrlich  and  Sachs  give  an  example  of  this.  The  amboceptor 
of  normal  goats  for  rabbit's  cells  and  that  obtained  by  injecting 
goats  with  ox  corpuscles  are,  of  course,  both  sensitized  with 
normal  goat  serum.  Now  if  we  take  rabbit's  corpuscles  and  add 
them  to  fresh  serum  (amboceptor-complement),  it  is  found  that 
both  the  complements  which  might  theoretically  be  present  are 
removed,  and  the  fluid  will  no  longer  reactivate  heated  immune 
serum  for  ox  corpuscles.  But  if  the  action  is  allowed  to  go  on  for 
a  short  time  only,  and  the  corpuscles  are  then  centrif  ugalized  down 
and  removed,  it  is  found  that  only  the  non-dominant  complement 


BACTERIOLYSIS    AND    ALLIED    PHENOMENA  155 

is  removed,  and  the  supernatant  fluid  will  still  reactivate  the  action 
of  heated  immune  serum  on  rabbit's  corpuscles.  This  is  not  the 
invariable  rule,  and  in  other  cases  the  non-dominant  complements 
are  not  seized  until  the  dominant  has  been  anchored.  In  either 
case  we  can  see  an  alternative  explanation  for  the  facts  observed 
by  Bordet  and  Gengou.  Ehrlich  points  out  that  the  presence  of 
the  power  to  bring  these  many  ferments  into  relation  with  the 
giant  molecule  of  the  protoplasm  must  be  of  advantage  in  cell 
nutrition,  as  enabling  the  greatest  possible  effect  to  be  produced, 
and  quotes  Hoffmeister  as  showing  that  the  liver  cell  contains  ten 
different  ferments,  and  Delbruck  as  attributing  five  to  the  yeast 
cell. 

The   discovery   that    the   haemolytic   action   of    snake   venom 
depends  on  the  mechanism  of  amboceptor  and  complement  made 


FIG.  36.— SHOWING  EFFECT  OF  ADDITION  OF  RABBIT'S  CORPUSCLES  TO  THE 
SERUM  SHOWN  IN  FIG.  35. 

The  amboceptor  unites  with  the  corpuscle,  and  all  the  complements  are  with- 
drawn. The  supernatant  fluid  loses  its  power  of  dissolving  ox  corpuscles 
previously  sensitized.  (See  also  Fig.  37.) 

by  Flexner  and  Noguchi,  and  the  excellent  subsequent  work  of 
Kyes,  made  us  realize  that  the  process  might  be  even  more 
complex.  Flexner  and  Noguchi  "^showed  that  snake  venom 
contains  two  substances,  one  of  which  is  thermostable,  not  being 
destroyed  at  90°  C.,  and  which  in  itself  has  no  power  of  bringing 
about  solution,  and  which  is  evidently  equivalent  to  amboceptor. 
It  may  be  reactivated  by  the  other  thermolabile  substance  or  by 
the  complements  of  normal  serum.  For  example,  horse  corpuscles 
can  be  dissolved  by  heated  venom  and  fresh  ox  serum,  ox  blood 
corpuscles  by  heated  venom  and  guinea-pig  serum,  and  so  on. 
Kyes  found  that  some  corpuscles,  washed  from  all  trace  of 
serum,  could  be  dissolved  by  cobra  venom  alone ;  others  required 
the  concomitant  presence  of  fresh  serum  of  the  same  or  other 
species.  Thus  the  corpuscles  of  man,  the  dog,  guinea-pig,  etc., 
are  dissolved  by  the  venom  alone,  whilst  those  of  the  ox,  sheep, 
and  goat  are  not.  He  formed  the  theory  (for  reasons  which  will 


HAEMOLYSIS — ENDO-COMPLEMENT 

not  be  discussed)  that  the  solution  of  corpuscles  without  the  action 
of  serum  was  due  to  a  complementing  of  the  venom  amboceptor 
by  means  of  complements  contained  in  the  corpuscle  itself,  which 
he  called  endo- complements.  This  he  proved  experimentally.  He 
argued  that  if  he  dissolved  the  red  blood-corpuscles  which  contain 
endo-complements  by  means  of  distilled  water,  the  solution  should 
act  as  though  it  contained  free  complements,  and  should  reactivate 
venom  amboceptor,  combined  with  corpuscles  which  it  was  unable 
to  dissolve  without  the  aid  of  serum.  This  was  found  to  be  the 
case.  A  laked  solution  of  human  corpuscles  mixed  with  venom 
would  haemolyze  the  corpuscles  of  the  ox,  sheep,  and  goat ;  but  a 
solution  of  ox  corpuscles  and  venom  would  not  dissolve  the  cor- 
puscles of  the  goat  or  sheep.  He  showed  that  these  endo- 
complements  are  thermolabile,  though  more  resistant  than  most 


FIG.  37. — SHOWING  EFFECT  OF  SHORT  EXPOSURE  OF  RABBIT'S  CORPUSCLES 
TO  THE  SERUM  SHOWN  IN  FIG.  35. 

The  non -dominant  complement  is  absorbed,  and  the  supernatant  fluid  no 
longer  dissolves  sensitized  ox  corpuscles,  whilst  retaining  its  action  on 
sensitized  rabbit  corpuscles.  (In  most  cases  the  dominant  complement  is 
removed  first.) 

of  those  in  sera.  They  are  destroyed  at  62°  C.  in  half  an  hour. 
Kyes  gave  an  additional  demonstration  of  his  theory  by  showing 
that  if  rabbit's  corpuscles  are  soaked  for  twenty-four  hours  in 
normal  saline  solution,  the  endo-complement  dissolved  out,  and 
could  be  demonstrated  in  the  fluid,  whilst  the  corpuscles  were  no 
longer  haemolyzed  by  snake  venom  without  the  addition  of  serum. 
He  holds,  therefore,  that  the  corpuscles  contain  endo-complements 
able  to  dissolve  them,  but  only  when  brought  into  organic  relation 
with  their  protoplasm  by  means  of  amboceptor. 

He  next  went  on  to  demonstrate  that  a  definite  chemical 
substance — lecithin — can  act  as  a  complement  to  snake  venom, 
and  that  either  it  or  the  serum-complement  could  act  as  a 
dominant  so  as  to  bring  about  solution,  or  that  the  two  might  act 
together.  Lastly,  he  was  able  to  prepare  a  combination  of  cobra 
venom  and  lecithin,  which  he  calls  cobra-lecithid,  which  is  quite 
different  in  its  physical  and  chemical  characters  from  either  of  its 


BACTERIOLYSIS    AND    ALLIED    PHENOMENA  157 

components,  and  which  acts  haemolytically  on  all  red  corpuscles. 
It  also  acts  much  more  quickly  than  ordinary  solutions  of  venom 
even  when  activated  with  lecithin,  and  it  is  very  thermostable, 
resisting  boiling  for  six  hours.  It  is  evidently  a  definite  chemical 
substance  and  of  great  theoretical  interest,  as  being  apparently 
an  amboceptor-complement  compound  preparable  in  a  pure  state. 
The  analogy  between  the  complements  and  the  exotoxins  leads 
to  the  inquiry  whether  the  production  of  an  anticomplement  occurs 
when  an  active  serum  is  injected  into  an  animal  of  another  species. 
Ehrlich  and  Bordet  both  proved  this  to  be  the  case.  He  injected 
fresh  horse  serum  into  goats,  and  obtained  a  serum  which  would 
prevent  the  normal  complementing  action  of  horse  serum.1  He 
easily  showed  that  this  was  not  due  to  any  action  which  it  exerted 
on  the  red  corpuscles  (rabbit's)  themselves  or  on  the  amboceptor. 


FIG.  38. — SHOWING  THE  EFFECT  OF  ADDING  TO  SENSITIZED  RABBIT'S 
CORPUSCLES  (a,  b)  A  MIXTURE  OF  FRESH  HORSE  SERUM  (CONTAINING 
COMPLEMENT,  c),  AND  SERUM  FROM  A  GOAT  WHICH  HAD  BEEN  INJECTED 
WITH  HORSE  SERUM  AND  CONTAINED  ANTICOMPLEMENT,  d. 

The  complement  and  anticomplement  combine,  and  the  corpuscles  are 
unaffected  ;  this  is  shown  by  the  fact  that  they  will  dissolve  on  the 
addition  of  fresh  complement. 

It  was  clear,  therefore,  that  it  acted  as  an  anticomplement.  The 
question  now  arose,  Was  this  action  exerted  on  the  haptophore  or 
the  zymophore  group  ?  The  following  experiment  shows  that  it 
acts  on  the  former  :  Sensitized  red  corpuscles  were  treated  with  a 
rmxture  of  normal  serum  and  of  serum  containing  anticomplement: 
no  solution  took  place.  The  corpuscles  were  then  centrifugalizecj. 
ott  andTresh  serum  added  :  haemolysis  occurred.  Evidently  the 
anticomplement  had  not  acted  on  the  zymophoric  group,  for  if  it 
had  the  haptophore  group  would  have  combined  with  the  ambo- 
ceptors  of  the  sensitized  corpuscles,  and  would  have  shielded 
them  from  further  action  when  fresh  serum  was  added.  What 

1  There  is  a  possible  fallacy  here,  to  be  mentioned  under  our  description  of 
Bordet  and  Gengou's  phenomenon.  It  may  be  simply  a  binding  of  the 
complement  in  the  precipitate.  This  is  discussed  later,  and  at  present 
Ehrlich's  views  will  be  set  out  as  if  they  were  definitely  proved. 


158    HAEMOLYSIS — ANTICOMPLEMENT    AND    COMPLEMENTED 

occurred  was  that  the  haptophore  radical  of  the  complement  had 
been  "  blocked  "  by  the  anticomplement. 

In  a  few  cases  anticomplements  which  will  act  on  some  only 
of  the  complements  of  a  sera  can  be  produced,  and  from  the  study 
of  these  additional  evidence  in  favour  of  the  multiplicity  of  com- 
plements has  been  adduced. 

As  regards  the  nature  of  these  anticomplements,  this  is  readily 
explicable  on  the  side-chain  theory.  Ehrlich  supposes  that  when 
a  foreign  serum  is  injected  into  an  animal  it  may  find  no  receptors 
for  which  it  has  an  affinity  ;  in  this  case  it  forms  no  anticomple- 
ment, and  such  cases  are  known  to  occur.  Or  it  may  find  receptors 
which  it  can  activate  completely,  just  as  the  normal  complement 
of  the  animal  can  do  ;  in  this  case  also  there  will  (under  ordinary 
circumstances)  be  no  formation  of  anticomplement.  Lastly,  it 
may  find  receptors  with  which  it  can  combine,  but  the  resulting 
combination  may  be  useless  in  the  nutrition  of  the  cell ;  in  this 


FIG.  39.— SHOWING  EFFECT  OF  ADDITION  TO  SENSITIZED  CORPUSCLES  OF  A 
MIXTURE  OF  COMPLEMENT  AND  COMPLEMENTOID. 

The  former  unite  with  the  complementophile  haptophore  groups  of  the  ambo- 
ceptor  ;  the  latter,  which  have  decreased  in  combining  affinity,  do  not. 

case  the  receptor  will  be  cast  off,  and  will  constitute  anti- 
complement. 

Lastly,  Ehrlich  was  able  to  show  that  in  some  cases  an 
auto-anticomplement  may  be  formed — i.e.,  an  antibody  which  can 
neutralize  the  complement  normally  present.  The  proof  for  this 
is  elaborate,  and  will  not  be  given  here. 

The  change  of  toxins  into  toxoids  is  paralleled  by  the  change  of 
complements  into  complementoids.  We  have  previously  spoken  of 
the  complements  as  being  destroyed  by  heat,  but  this  is  not  quite 
correct ;  they  lose  their  zymophore  groups,  but  retain  their  hapto- 
phore portions,  though  these  appear  to  lose  some  of  their  combining 
affinity.  The  proof  of  the  existence  of  complementoids  is  simple  : 
when  injected  into  animals  they  call  forth  the  production  of 
anticomplements,  just  as  toxoids  call  forth  the  production  of 
antitoxin. 

For  some  time  it  was    found    impossible  to   demonstrate  the 


BACTERIOLYSIS    AND    ALLIED    PHENOMENA  159 

presence  of  complementoids  in  test-tube  experiments,  and  this 
was  owing  to  the  fact  that  the  affinity  of  the  haptophore  group  of 
the  complements  suffered  loss  during  the  process  of  heating,  so 
that  when  a  mixture  of  complement  and  complementoid  was 
added  to  sensitized  red  corpuscles,  the  former  combined  just  as 
well  as  if  the  latter  were  not  present.  "  Blocking  "  of  the  com- 
plementophile  group  of  the  amboceptor  appeared  not  to  occur  ; 
but  Ehrlich  and  Sachs  were  able  subsequently  to  demonstrate 
such  a  phenomenon  in  this  wise  :  Dog  serum  contains  an  ambo- 
ceptor for  guinea-pig's  corpuscles,  and  this  can  be  activated  either 
by  dog's  or  guinea-pig's  serum.  New  when  a  mixture  of  heated 
dog's  serum,  guinea-pig's  corpuscles,  and  guinea-pig's  serum  was 
made,  solution  took  place ;  but  when  the  corpuscles  and  heated 
serum  were  added  together,  allowed  to  stand,  and  then  centrifuga- 
lized  and  removed,  no  solution  occurred  on  the  addition  of  fresh 


FIG.  40.— DEMONSTRATION  OF  "  BLOCKING  "  OF  COMPLEMENTOPHILE  GROUPS 
BY  COMPLEMENTOIDS. 

a  =  complementoid,    and   &  =  amboceptor   in   heated   dog   serum;    r  =  rabbit's 
corpuscles  (see  text). 

guinea-pig's  serum.  Ehrlich  proved1  that  the  combination  of 
amboceptor  and  corpuscles  took  place  as  usual,  and  that  it  was  then 
followed  by  a  blocking  of  the  free  arms  of  these  amboceptors  by 
the  complementoids  present  in  the  heated  dog's  blood.  In  this 
case,  therefore,  the  complement  had  not  suffered  any  appreciable 
change  in  combining  capacity  as  a  result  of  its  conversion  into 
complementoid. 

It  follows,  therefore,  that  when  we  heat  fresh  serum,  or  allow  it 
to  stand  a  few  days  at  the  room  temperature,  we  do  not  get  rid 
of  the  complements  altogether.  This  can  be  done,  as  shown  by 
Von  Dungern  and  others,  by  shaking  the  serum  with  cells  (liver, 
kidney,  etc.),  yeast,  fungi,  certain  bacteria,  powdered  charcoal, 
etc.  This  abstraction  of  complements  by  inert  bodies  is  a  non- 
specific process  entirely  unlike  its  fixation  in  haemolysis,  etc., 

1  The  proof  is  complete  and  conclusive,  but  too  long  to  give  here. 


i6o 


HAEMOLYSIS — ANTI  AMBOCEPTOR 


and  Ehrlich  compares  it  to  a  process  of  physical  absorption.  The 
presence  of  the  phenomenon  must  be  borne  in  mind,  since  it  must 
make  us  careful  how  we  interpret  the  mere  disappearance  of 
complement  from  a  fluid  after  it  has  acted  on  articulate  sub- 
stances. The  true  test  of  specificity  is  its  action  on  these 
substances,  not  its  absorption  by  them.  The  yeast  cells  are  in  no 
ways  injured  through  having  absorbed  complement. 

We  have  not  yet  finished  our  study  of  the  complements,  but  it 
will  be  convenient  to  continue  it  later,  and  to  conclude  here  Ehrlich's 
studies  on  the  antilysins.  Anticomplement  has  been  already 
discussed,  and  we  will  now  turn  to  anti-amboceptor. 

This  is  a  substance  of  nature  different  to  any  that  we  have 
previously  studied,  in  that  it  is  an  antibody  to  an  antibody.  It  is 
prepared  by  injecting  an  immune  serum  into  an  animal  other  than 
that  from  which  it  was  derived.  Thus,  if  an  antityphoid  (bacterio- 
lytic)  serum  procured  by  immunizing  a  horse  with  typhoid  bacilli 
be  injected  into  a  rabbit,  the  serum  of  the  latter  acquires  the 
power  of  neutralizing  the  bacteriolytic  action  of  the  former. 
Antibodies  to  antibodies  cannot  always  be  produced;  thus,  anti- 
antitoxin  is  not  yet  known.  Again,  Metchnikoff  was  unable  to 
prepare  antispermotoxin  by  injecting  a  cytolytic  serum  for  sperma- 
tozoa into  the  guinea-pig.  In  this  case  there  can  be  no  question  of 
the  absence  of  suitable  receptors,  for  the  animal's  spermatozoa  are 
attacked  in  vitro  by  the  serum  injected.  Hence  Ehrlich  made 
the  assumption  of  the  presence  of  sessile  receptors,  which  are  not 
cast  off  when  the  suitable  antibody  is  injected. 

In  studying  the  anti-amboceptor  concerned  in  haemolysis, 
Ehrlich  first  prepared  a  haemolytic  serum  by  injecting  a  rabbit 
with  ox  blood.  When  the  serum  of  the  latter  was  found  to  be 
powerfully  haemolytic,  it  was  injected  subcutaneously  into  a  goat : 
120  c.c.  was  given  during  an  interval  of  two  months.  At  the  end 
of  this  time  this  goat  serum  was  powerfully  antihaemolytic.  This 
was  shown  as  follows  :  0-00125  c.c.  of  (immune)  rabbit  serum  was 
found  to  haemolyze  completely  i  c.c.  of  a  5  per  cent,  emulsion  of 
ox  corpuscles,  provided  that  sufficient  normal  guinea-pig  serum 
is  added  to  provide  complement.  Now  three  times  this  amount 
(0-00375),  plus  0-5  of  the  goat  serum,  was  added  to  i  c.c.  of  the 
emulsion  of  corpuscles,  allowed  to  act  at  40°  C.  for  an  hour,  centri- 
fugalized,  and  0-15  c.c.  of  normal  guinea-pig  serum  (complement) 
added  to  the  corpuscular  sediment.  No  solution  took  place,  so 
that  0-5  c.c.  of  the  anti-antiserum  had  completely  neutralized  three 


BACTERIOLYSIS    AND   ALLIED    PHENOMENA  l6l 

dissolving  doses  of  the  antiserum.     A  control  test  showed  that 
normal  goat  serum  was  without  action. 

Theoretically,  two  anti-amboceptors  are  possible :  one  which 
will  combine  with  the  cytophile  group  of  the  amboceptor,  and  one 
which  will  seize  on  the  complementophile.  In  his  earlier  studies 
Ehrlich  thought  that  the  former  was  that  actually  produced  when 
immune  sera  are  injected  into  animals  of  another  species.  He 
later  altered  his  opinion,  and  now  holds  that  the  substance 
actually  formed  is  an  antibody  to  the  complementophile  hapto- 
phore  of  the  amboceptor.  He  was  led  to  this  conclusion  by  a 
discovery  of  Bordet's,  which  was  adduced  as  evidence  against  the 
amboceptor  theory,  but  which  was  ingeniously  adapted  to  its 
defence  by  Ehrlich.  The  discovery  was  as  follows :  Normal 
rabbit  serum  when  injected  into  a  guinea-pig  leads  to  the  production 
of  an  anti-antibody,  which  neutralizes  the  haemolysin  formed  by 


FIG.    41. — THE    COMPLEMENTOPHILE    HAPTOPHORE    GROUPS   OF    AN    AMBO- 
CEPTOR "BLOCKED"  BY  ANTIAMBOCEPTOR  (a,  a}. 

immunizing  with  ox  blood.  This,  of  course,  is  readily  explicable 
if  normal  rabbit  serum  contained  an  amboceptor  for  ox  corpuscles, 
but  this  is  not  the  case.  Ehrlich  argued  as  follows :  An  anti- 
amboceptor  is  obviously  formed,  but  the  normal  rabbit  serum 
injected  contains  no  amboceptor — i.e.,  no  cytophile  groups  ;  hence 
the  ant  -amboceptors  produced  must  be  anticomplementophile. 
For  this  to  happen  it  is  necessary  that  this  normal  rabbit  serum 
must  contain  complementophile  groups,  and  this  is  obviously  the 
case,  since  other  amboceptors,  each  with  a  complementophile 
group,  are  present.  Therefore,  on  the  assumption  that  these 
groups  are  similar,  even  when  they  occur  in  different  amboceptors, 
the  case  is  clear.  But  are  these  complementophile  groups  similar  ? 
Does  not  this  contradict  Ehrlich's  views  on  the  multiplicity  of 
complements  ?  To  avoid  this  difficulty,  Ehrlich  falls  back  on  his 
polyceptor  theory.  All  or  most  amboceptors  are  really  poly- 
ceptors,  differing  only  or  mainly  in  their  cytophile  groups,  and 
possessing  numerous  complementophile  groups,  which  are  similar 
or  identical  in  different  cases  in  the  same  species.  But  if  this 

ii 


l62  RECAPITULATION    OF   EHRLICH'S   THEORIES 

is  the   case,  anti-amboceptors  should  be  non-specific ;  they  are 

directed  against  the  complementophile  groups,  which  are  the  same 

in  all  amboceptors  from  the  same  species.     This  subject  has  not 

yet  been  fully  investigated,  but  Pfeiffer  and   Friedberger  have 

shown  that  the  antibody  to  the  immune  body  against  the  cholera 

vibrio  also  acts   against   the   antityphoid   serum.      Ehrlich   also 

showed  that  if  anti-amboceptor  be  added  to  sensitized  cells^these 

are  protected  against  the   subsequent   addition   of   complement. 

/This,  of  course,  is  attributable  to  the  fact  that  the  anti-amboceptor 

I  unites  with  the  free  complementophile  groups,  and  "  blocks  "  them 

[^against  the  subsequent  access  of  the  complement  molecule. 

Ehrlich  leaves  undecided  the  question  as  to  whether  an  anti- 
cytophilic  anti-amboceptor  is  ever  produced,  but  holds  that  it 
might  be  formed  if  by  any  process  we  could  destroy  the  cytophile 
haptophore  of  the  amboceptor  molecule.  Under  ordinary  circum- 
stances the  complementophile  group  has  a  greater  affinity  for  its 
cell  receptor  than  has  the  cytophile  for  the  receptors  with  which 
it  could  combine ;  union  takes  place  between  the  former,  and  the 
latter  is,  so  to  speak,  pulled  out  of  the  way  of  its  affinity. 

It  is  now  advisable  to  recapitulate  briefly  some  of  the  main 
points  in  Ehrlich's  theory  of  the  structure  of  the  bacteriolytic  and 
haemolytic  sera.  Firstly,  as  regards  complements :  Of  these  there  is 
very  great  number,  and  each  is  especially  adapted  for  the  solution  of 
one  or  more  varieties  of  cells,  which  it  can  dissolve  in  the  presence 
of  a  suitable  amboceptor  ;  it  is  known  as  the  dominant  comple- 
ment. Other  complements,  however,  may  help  in  the  process, 
and  these  are  termed  non-dominant.  In  general  terms  the  comple- 
ments, which  are  especially  active  in  haemolysis,  have  but  little 
action  on  bacteria,  and  vice  versa.  Secondly,  as  regards  the  ambo- 
ceptor :  This  is  really  a  polyceptor,  and  is  so  constituted  that  it 
can  combine  with  the  cell  to  be  dissolved,  on  the  one  hand,  and 
with  a  large  number  of  molecules  of  complement,  on  the  other. 

Of  the  further  complications  of  the  theory  which  Ehrlich  has 
introduced  to  explain  new  phenomena  as  they  arise,  of  the 
amboceptoids,  of  loose  and  firm  union,  etc.,  we  do  not  propose 
to  speak.  That  the  theory  needs  these  complications  in  order  to 
account  for  the  phenomena  is  not  necessarily  in  its  disfavour,  for 
the  phenomena  themselves  are  complex  in  the  extreme.  And  it 
must  be  regarded  as  a  strong  argument  in  its  favour  that  Ehrlich 
has  again  and  again  deduced  results  from  this  theory  which  sub- 
sequent research  has  shown  to  be  correct ;  and  very  few  hypo- 


BACTERIOLYSIS   AND   ALLIED    PHENOMENA  163 

theses  can  be  adduced  which  have  been  so  rich  in  leading  to  the 
discovery  of  new  facts. 

It  will  now  be  necessary  for  us  to  glance  briefly  at  some 
alternative  hypotheses,  and  it  must  be  pointed  out  that  the  com- 
paratively short  notice  they  will  receive  must  not  be  taken  to 
imply  that  there  is  less  to  be  said  in  their  favour.  The  trend  of 
modern  research  tends  on  the  whole  against  Ehrlich's  views ;  yet 
in  the  history  of  immunity  his  researches  will  always  be  regarded 
as  of  the  greatest  interest  and  value. 

It  may  be  pointed  out  that  the  cast-off  receptors  of  red 
corpuscles,  bacteria,  etc.,  may  combine  with  the  complemento- 
phile  groups  of  an  amboceptor,  and  thus  appear  to  act  as  a 
cytophilic  anti-amboceptor. 

Bordet  holds  that  the  immune  body  is  not  an  amboceptor  at 
all — i.e.,  that  it  does  not  act  as  a  link  between  the  corpuscle  or 
bacterium  and  the  complement,  but  that  it  sensitizes  the  former 
and  renders  it  susceptible  to  the  action  of  the  latter.  On  Ehrlich's 
theory,  amboceptor  unites  with  cell  and  complement  with  ambo- 
ceptor;  on  Bordet's,  both  substances  unite  with  the  cell  direct. 
On  the  latter  theory  many,  if  not  all,  of  the  results  observed  by 
Ehrlich  and  others  are  explicable,  and  there  appears  to  be  no 
experimentum  crucis  by  which  the  truth  can  be  determined.  A  diffi- 
culty in  the  way  of  accepting  Ehrlich's  amboceptor  theory  is  this : 
there  is  no  proof  that  complement  and  immune  body  ever  unite 
unless  the  latter  has  already  combined  with  a  cell,  or  with  anti- 
amboceptor,  or  with  free  receptors.  The  only  example  to  the 
contrary  is  supplied  by  Ryes'  cobra-lecithid,  which,  though  of 
great  interest,  can  hardly  be  quoted  as  a  case  in  point,  since 
lecithin  differs  so  markedly  from  the  ordinary  thermolabile 
complements  of  serum.  Another  case  (in  the  deviation  of  the 
complements)  in  which  this  process  appears  to  take  place  is 
extremely  complicated,  and  the  evidence  in  favour  of  the  direct 
union  of  complement  and  antibody  quite  unsatisfactory.  It 
follows,  then,  that  we  must  either  assume  that  the  mere  union 
of  the  cytophile  group  of  the  antibody  with  some  other  object 
increases  the  affinity  of  the  complementophile  group  for  comple- 
ment (as  has  been  done  here),  or  we  must  agree  with  Bordet  that 
cell  and  complement  unite  directly,  but  only  after  the  latter  has 
been  prepared  by  the  action  of  immune  body.  A  very  important 
observation  of  Muir's  is  of  interest  in  this  connection.  Muir 
showed  that  after  red  corpuscles  had  been  saturated  with  immune 

II — 2 


164  HAEMOLYSIS — BORDET'S  VIEWS 

body  and  then  with  complement,  some  of  the  immune  body,  but 
none  of  the  complement,  might  be  dissociated  from  the  combina- 
tion, and  become  free  in  the  fluid.  It  is  difficult,  though  perhaps 
not  quite  impossible,  to  reconcile  this  experiment  with  Ehrlich's 
theory,  whilst  it  is  really  explicable  on  that  of  Bordet ;  but  Muir 
(whose  recent  work  on  this  difficult  point  should  be  consulted) 
has  not  come  to  a  definite  conclusion  as  to  the  nature  of  the 
combination. 

Bordet  also  holds  peculiar  views  on  the  subject  of  the  union 
of  corpuscles  and  immune  body.  According  to  Ehrlich,  the  com- 
bination is  due  to  a  strong  chemical  combining  affinity  between 
the  two  substances,  and  the  resulting  compound  is  a  stable  one, 
which  is  not  readily  dissociable.  Cells  thus  activated  possess  a 
strong  affinity  for  complement,  and  the  whole  process  is  a  chemi- 
cal one,  dependent  on  ordinary  chemical  laws  and  obeying  the 
laws  of  multiple  proportions.  This  was,  on  the  whole,  confirmed 
by  Muir,  who  proved  that  corpuscles  combined  with  multiple 
doses  of  immune  body  took  up  multiple  doses  of  complement ; 
but  he  also  showed  that  the  corpuscle-antibody  compound  did 
dissociate,  so  that  when  corpuscles  which  had  been  saturated 
with  immune  body  were  placed  in  contact  with  normal  corpuscles, 
some  of  the  amboceptor  left  the  sensitized  and  attached  them- 
selves to  the  normal.  Bordet  adduced  evidence  to  show  that  the 
combination  is  of  a  remarkable  nature,  and  dependent  on  obscure 
chemico-physical  reactions.  He  took  a  sample  of  haemolytic 
serum,  and  determined  the  amount  of  blood  which  it  could 
haemolyze  when  the  two  were  mixed  together.  We  may  assume, 
on  Ehrlich's  theory,  that  all  the  suitable  receptors  in  the  cor- 
puscles were  saturated  with  amboceptor.  But  Bordet  showed 
that  if  the  corpuscles  were  added  a  little  at  a  time,  a  very  much 
smaller  dose  might  take  up  all  the  immune  body.  Hence  he 
compared  the  process  to  the  staining  of  filter-paper  when  im- 
mersed in  a  dye.  If  the  paper  be  added  at  once,  it  will  be  stained 
a  uniform  colour,  whereas  if  it  be  added  a  little  at  a  time,  the  first 
pieces  will  be  stained  deeply,  the  subsequent  ones  less  and  less, 
until  the  dye  is  completely  absorbed.  It  would  appear  to  explain 
the  phenomenon  equally  well  if  we  assumed  that  the  corpuscles 
could  be  haemolyzed  by  fewer  amboceptors  than  they  could  take 
up,  and  there  is  some  experimental  evidence  of  this.  Thus  Muir 
showed  that  after  red  corpuscles  have  been  dissolved  by  haemo- 
lytic  sera,  the  addition  of  more  immune  body  will  lead  to  the 


BACTERIOLYSIS    AND    ALLIED    PHENOMENA  165 

absorption  of  more  complement.  It  may  be  added,  however,  that 
many  of  the  recent  researches  on  antibodies  point  to  them  as 
being  governed  by  the  very  complex  laws,  as  yet  not  fully  under- 
stood, which  regulate  the  actions  of  colloids  on  one  another 
and  on  crystalloids.  This  is  discussed,  though  very  briefly,  in  a 
subsequent  chapter. 

As  regards  the  evolutionary  significance  of  the  facts  of  cytolysis 
(using  the  word  to  cover  the  solution  of  bacterial  and  all  other 
cells),  there  are  two  theories  which,  though  apparently  widely 
different,  have,  on  ultimate  analysis,  much  in  common.  We  have 
seen  how  Ehrlich  explains  the  process  of  cell  nutrition,  the  role 
which  he  attributes  to  complement,  and  the  method  in  which  he 
conceives  the  complex  antibodies  to  be  formed.  On  his  theories, 
therefore,  the  fundamental  process  is  one  of  cell  nutrition,  and 
we  may  imagine  it  to  have  become  adapted  to  serve  as  a  means 
of  immunity  during  the  course  of  natural  selection :  the  animals 
which  could  make  use  of  their  mechanism  of  cell  nutrition  as  a 
means  of  defence  against  invading  micro-organisms  would  survive 
and  perpetuate  their  species ;  the  others  could  not. 

We  may  add  a  few  more  considerations  on  this  process  of 
cell  nutrition.  We  must  assume  that  the  food  molecules  of 
proteid  which  circulate  in  the  blood  are  in  an  indifferent  form, 
and  are  equally  well  adapted  for  the  nourishment  of  cells  of  the 
brain,  liver,  etc.,  this  method  of  transmission  of  nourishment 
having  been  found  the  most  convenient  and  economical.  The 
first  requisite  for  the  nutrition  of  the  cell  is  that  one  of  these 
molecules  shall  be  brought  into  close  relations  with  its  protoplasm. 
Ehrlich  speaks  of  this  as  being  due  to  the  selective  action  of  the 
cell  receptors,  but  we  must  not  be  misled  by  terms  or  diagrams, 
however  convenient  they  may  be  to  enable  us  to  form  a  mental 
picture  of  the  process.  Ehrlich  simply  means  that  certain 
specialized  molecules  or  groups  of  molecules  have  a  chemical 
combining  affinity  for  certain  food  molecules.  This  union  is  the 
first  requisite,  and  in  some  cases  it  may  be  sufficient,  and  the 
molecule  may  be  forthwith  incorporated  with  the  cell  protoplasm. 
In  most  cases,  however,  this  is  not  so,  and  the  cell  has  to 
transform  an  indifferent  molecule  of  proteid  material  into  a  form 
suitable  for  assimilation  into  itself.  We  now  know  that  most,  if 
not  all,  of  the  activities  of  a  living  cell  in  metabolism  are  dis- 
charged through  the  agency  of  enzyme  action,  and  that  each  of 
the  metabolic  actions  of  a  cell  appears  to  depend  on  a  special 


l66  CELL   NUTRITION 

enzyme.     It  is  natural,  therefore,  that  the  cell  should  make  use  of 
soluble  ferments  in  preparing  the  molecule  which  it  has  seized. 

We  now  (thanks  to  the  researches  of  Fischer,  Hierf elder,  and 
others)  know  that  the  action  of  these  enzymes  is  strictly  specific, 
and  dependent  on  a  stereo-chemical  relation  between  the  ferment 
and  the  body  acted  on,  and  that  it  is  necessary  in  all  cases  for  a 
union  to  be  brought  about  between  the  two  substances  before  the 
specific  action  is  effected.  Thus,  if  the  two  could  unite  in  the  blood, 
the  molecules  would  be  broken  down  before  they  reach  the  cells,  and 
the  food  material  would  reach  it  in  an  indifferent  form,  which, 
however  well  adapted  to  some  cells,  might  be  useless  to  others. 
The  substances,  therefore,  do  not  unite  in  the  blood-stream  at  all, 
but  the  cell  first  seizes  a  molecule  of  food-stuff,  and  then  one  or 
more  ferments  which  can  change  it  into  the  exact  form  which  the 
cell  requires.  Thus,  the  process  of  cell  nutrition,  according  to 
Ehrlich,  consists  in  the  establishment  of  a  link  between  the  food 
and  a  suitable  ferment  after  the  former  has  been  selected  by  the 
cell. 

We  may  inquire  why  the  cell  does  not  form  its  own  ferments. 
In  certain  cases  it  may  do  so ;  the  existence  of  the  endocom- 
plements  lends  support  to  this  view.  But  it  is  readily  conceivable 
that  it  may  be  more  economical  in  every  way  for  the  animal 
to  limit  this  process  of  enzyme  formation  to  certain  specialized 
cells  or  tissues,  just  as  pepsin  is  formed  by  the  cells  of  the  gastric 
glands.  This  is  probably  the  case,  and  all  experience  goes  to 
show  that  the  cells  to  which  this  duty  is  delegated  are  the 
leucocytes. 

Ehrlich,  therefore,  conceives  of  the  cytotoxic  mechanism  of 
amboceptors  and  complements  as  being  adapted,  in  the  first  place, 
to  cell  nutrition,  and,  secondarily,  to  the  defence  of  the  body  by 
bringing  about  solution  of  foreign  cells.  Metchnikoff 's  views  may 
be  summarized  as  follows:  He  holds  that  both  the  substances 
taking  part  in  cytolysis  are  ferments,  and  that  both  are  adapted 
for  intracellular  digestion.  His  views  on  phagocytosis  will  be 
discussed  subsequently,  and  here  it  will  be  sufficient  to  give  the 
merest  outline.  He  has  shown  that  in  the  lowest  Metazoa — e.g., 
in  the  Coelenterata — digestion  is  an  intracellular  process  entirely  ; 
the  hypoblastic  cells  lining  the  alimentary  canal  seize  food  particles 
from  the  lumen  and  digest  them,  but  form  no  secretion  like  those 
of  the  higher  animals.  In  the  cells  which  have  seized  and  are 
digesting  food  particles  in  this  way  he  was  able  to  demonstrate 


BACTERIOLYSIS    AND   ALLIED    PHENOMENA  167 

ferments  allied  to  pepsin  or  trypsin,  which  had  evidently  been 
formed  to  digest  the  ingested  food  material.  He  then  showed 
the  importance  of  this  process  in  the  absorption  of  cells,  etc.,  in 
the  tissues  of  higher  animals,  and  demonstrated  that  the  two 
processes  are  altogether  similar.  Hence  his  views  on  the  nature 
of  the  cytolysins  arise  easily  and  naturally.  He  holds  that  com- 
plement, or,  as  he  calls  it,  cytase,  is  the  digestive  secretion  of 
the  leucocytes,  and  that,  under  ordinary  circumstances,  it  is 
retained  within  the  leucocytes  ;  it  is  only  set  free  when  leucocytes 
are  dissolved  (phagolysis),  either  as  the  result  of  an  injection  of  a 
foreign  substance  or  in  the  process  of  clotting  (this  theory,  as  we 
shall  show,  is  held  by  many  other  authorities).  Thus  in  Pfeiffer's 
phenomenon  the  first  result  of  the  injection  is  phagolysis,  and  the 
ferments  set  free  in  the  process  immediately  attack  the  cholera 
vibrios.  Metchnikoff  has  proved  clearly  that  the  injection  of 
almost  anything  into  the  peritoneal  cavity  leads  to  a  diminution  in 
the  number  of  leucocytes,  but  the  proof  of  the  existence  of 
phagolysis  under  these  circumstances  is  less  convincing.  Cytase, 
therefore,  is  a  digestive  ferment  adapted  to  deal  with  large  masses 
of  food  substance  rather  than  with  molecules,  as  Ehrlich  supposes, 
and  is  normally  intracellular,  being  formed  especially  for  intra- 
cellular  digestion. 

Metchnikoff  sees  in  amboceptor,  or  fixator,  a  substance  alto- 
gether analogous  to  enterokinase,  and  acting,  like  it,  as  an 
accessory  digestive  ferment,  which  has  for  its  object  the  linking 
of  the  more  potent  ferment  to  the  food  to  be  digested.  He 
regards  it  also  as  being  formed  in  the  leucocytes,  and  for  this 
reason  (amongst  others,  which  will  be  enumerated  subsequently)  : 
He  states  that  the  amount  of  fixator  or  amboceptor  produced 
is  proportional  to  the  amount  of  phagocytosis  which  occurs  during 
the  absorption  of  the  antigen.  For  example,  he  states  that  when 
defibrinated  goose  blood  is  injected  into  the  guinea-pig  sub- 
cutaneously  there  is  but  little  phagocytosis,  the  blood  being 
dissolved  extracellularly,  and  but  little  immune  body  is  produced  ; 
but  when  the  injection  is  made  into  the  peritoneum  there  is  much 
phagocytosis  and  much  development  of  antibody.  There  is  cer- 
tainly a  remarkable  difference  between  the  various  tissues  as 
origins  of  antibodies,  and,  as  a  rule,  the  subcutaneous  and  con- 
nective tissues  are  most  potent  in  this  respect,  the  peritoneum 
next,  and  the  circulating  blood  worst ;  but  there  is  no  sufficient 
evidence  to  show  that  this  depends  on  the  amount  of  phagocytosis 


168  METCHNIKOFF'S  VIEWS 

which  occurs  in  these  different  situations.  The  site  of  the  forma- 
tion of  an  immune  body  will  be  referred  to  again. 

Thus,  whilst  Ehrlich  sees  in  the  substances  taking  part  in 
cytolysis  evidence  of  the  nutrition  of  the  living  molecules  of  cell 
protoplasm,  Metchnikoff  sees  in  them  ferments  which  were  also, 
in  their  first  appearance  in  the  animal  economy,  designed  for  the 
elaboration  of  the  nourishment  of  the  body  or  of  certain  cells 
therein,  the  difference  being  that,  according  to  him,  their  primitive 
function  was  the  digestion  of  large  particles  as  well  as  of  molecules. 
But  in  the  highly-organized  animals  with  which  we  have  chiefly 
to  deal  this  function  has  been  changed,  for  in  them  phagocytes  do 
not  ingest  bacteria  and  red  corpuscles  for  the  sake  of  the  nourish- 
ment they  contain,  but  to  rid  the  body  of  invaders ;  the  body  as  a 
whole  is  nourished  through  the  agency  of  other  extracellular 
digestive  ferments,  which  are  secreted  into  the  alimentary  canal. 
It  follows,  therefore,  that  cytase  is  unnecessary  in  the  circulating 
blood,  and,  on  this  supposition,  does  not  usually  exist  in  that  situa- 
tion. Metchnikoff  admits  that  immune  body  does  so  exist — and  has 
indeed  supplied  a  remarkable  proof  of  the  fact,  since  he  showed 
that  the  spermatozoa  of  immunized  guinea-pigs  are  combined 
with  immune  body,  and  only  need  the  addition  of  fresh  serum  for 
cytolysis  to  occur.  Cytase  and  fixator,  on  MetchnikorPs  explana- 
tion of  the  phenomena,  must  be  regarded  as  substances  which  in 
the  evolution  of  the  animal  kingdom  were  first  developed  as 
digestive  juices,  but  which  now  are  entirely  subservient  to  the 
defence  of  the  body  against  invaders.  According  to  Ehrlich,  they 
are  both  in  daily  use  in  nourishment,  and  their  defensive  function 
is  of  less  importance  than  their  value  in  cell  nutrition.  In  either 
case,  the  conception  of  immunity,  as  being  fundamentally  a  process 
of  nutrition,  is  a  most  striking  one. 

We  have  now  to  turn  to  a  phenomenon  which  is  of  the  highest 
theoretical  importance,  and  which  bids  fair  to  be  of  great  practical 
value  in  diagnosis.  We  have  already  referred  to  the  fact  that 
Bordet,  the  chief  advocate  of  the  Unitarian  theory  of  complement, 
showed  that  if  red  corpuscles  be  sensitized  with  amboceptor  and 
added  to  fresh  serum,  the  latter  is  deprived  of  all  complementary 
activity.  The  same  phenomena  occur  with  sensitized  bacteria. 
When,  for  instance,  typhoid  bacilli  which  had  been  acted  on  by 
heated  typhoid  serum  were  added  to  fresh  blood,  allowed  to  act,  and 
then  centrifugalized,  it  was  found  that  the  supernatant  fluid  had  no 
longer  the  power  of  dissolving  red  corpuscles  sensitized  by  suitable 


BACTERIOLYSIS    AND    ALLIED    PHENOMENA  l6g 

amboceptor.  These  facts  are  admitted  by  Ehrlich,  but  he  shows 
that  they  do  not  constitute  any  real  evidence  against  the  pluralist 
conception,  since  in  some  cases  it  may  be  shown  that,  though  all 
complements  are  absorbed,  this  may  be  at  different  rates,  and  by 
stopping  the  process  at  a  proper  time,  some  may  have  disappeared, 
whilst  others  are  left. 

The  practical  importance  of  these  observations  arises  from  the 
fact  that  they  give  us  a  method  by  which  we  can  demonstrate  the 
presence  or  absence  of  an  antibody  to  a  given  antigen,  or  of  an 
antigen  to  a  given  antibody.  For  example,  the  sensibilatrice  or 
amboceptor  for  the  tubercle  bacillus  is  very  difficult  to  demonstrate, 
since  the  organism  is  so  resistant  that  it  is  never  obviously  dis- 
solved, even  partially,  in  the  most  potent  serum  we  can  obtain. 
Nor  are  bactericidal  experiments  more  promising,  owing  partly  to 
the  resisting  power  of  the  organism  and  partly  to  the  technical 
difficulties.  The  only  evidence  (apart  from  the  presence  of 
agglutinins)  which  we  have  in  favour  of  the  formation  of  specific 
antibodies  to  tuberculosis  is  derived  from  an  application  of  Bordet's 
phenomenon.  The  experiments  were  carried  out  as  follows :  A 
guinea-pig  was  injected  with  the  bacillus  of  avian  tuberculosis,  to 
which  it  is  but  slightly  sensitive,  and  the  blood  was  examined  by 
mixing  it  with  an  emulsion  of  the  bacilli.  If  amboceptors  were 
present  they  would  combine  with  the  bacilli,  and  draw  to  them  all 
the  complements  of  the  fluid,  which  would  thus  lose  its  power  of 
activating  suitably  sensitized  red  corpuscles.  This  was  found  to 
occur,  and  Bordet  and  Gengou  deduced  that  the  guinea-pig  had 
formed  antibodies  to  the  slightly  virulent  tubercle  bacilli.  When, 
on  the  other  hand,  the  guinea-pigs  were  injected  with  virulent 
human  tubercle  bacilli,  no  such  antibodies  could  be  demonstrated. 

As  an  example  of  the  recognition  of  an  antigen  by  means  of 
Bordet's  phenomenon,  we  shall  quote  Bruck's  demonstration  of 
the  presence  of  tuberculin,  or  at  least  of  some  derivative  of  the 
tubercle  bacillus,  in  the  blood  of  patients  suffering  from  general 
tuberculosis.  Here  the  problem  is  changed.  We  have  a  specimen 
of  serum  which  we  want  to  test,  not  for  the  antibody,  but  for  the 
antigen.  The  procedure  is  as  follows  :  The  serum  is  heated  and 
mixed  with  a  serum  known  to  contain  antibodies  to  the  tubercle 
bacillus  (antituberculin  of  Hochst).  To  this  mixture  is  added 
fresh  guinea-pig's  serum,  and  lastly  sensitized  red  corpuscles.  It 
is  found  that  no  haemolysis  takes  place.  In  a  control  experiment, 
in  which  normal  human  blood  took  the  place  of  that  from  the 


170  THE    BORDET-GENGOU    PHENOMENON 

patient,  haemolysis  occurred ;  it  followed,  therefore,  that  the 
patient's  blood  contained  derivatives  of  the  tubercle  bacillus,  or 
(as  we  may  fairly  say)  its  toxins. 

The  technique  of  these  experiments  is  somewhat  difficult,  but 
the  results  have  been  so  important,  and  the  method  seems  so 
likely  to  play  a  part  of  importance  in  clinical  diagnosis,  that  it  will 
be  discussed  in  a  further  chapter. 

Gengou's  phenomenon  is  similar  to  Bordet's.  It  requires  a 
little  anticipation  of  facts  to  be  discussed  subsequently  concerning 
the  precipitins.  These  are  antibodies  obtained  by  the  injection 
of  proteid  solutions,  and  having  the  power  of  uniting  with  these 
proteids  to  form  insoluble  precipitates.  Gengou  showed  that  in 
this  combination  of  antigen  and  antibody  the  same  absorption  of 
complement  occurred  as  in  the  case  of  sensitized  red  corpuscles 
or  bacteria.  The  process  is  an  extraordinarily  delicate  one,  and 
it  has  been  shown  to  be  demonstrable  with  as  little  as  o-oooooi  c.c. 
of  the  antigen  (in  this  case  normal  human  serum),  and  may  occur 
when  the  serum  used  is  so  dilute  that  no  visible  precipitation 
occurs. 

It  appears,  further,  that  this  process  of  fixation  of  complement 
is  a  general  one,  occurring  whenever  an  antigen  and  its  antibody 
unite,  whether  the  antigen  occurs  in  solid  or  liquid  form,  and 
whether  the  resulting  compound  forms  a  precipitate  or  remains  in 
solution.  It  has  been  shown  by  Nicolle  and  by  Armand-Delille 
to  occur  in  the  neutralization  of  tetanus  and  diphtheria  toxins  by 
their  appropriate  antitoxins,  and  by  Pozerski  in  the  interaction  of 
papain  and  its  antiferment.  It  has  been  shown  by  Guedini  that 
when  hydatid  fluid  is  mixed  with  the  serum  of  an  animal  which 
has  been  injected  therewith  a  similar  phenomenon  occurs,  and 
this  fact  has  been  suggested  and  applied  by  Weinberg,  Parvu,  and 
Lanbry  to  the  diagnosis  of  hydatid  cysts  in  man. 

The  theoretical  importance  of  this  phenomenon  arises  from  the 
light  which  it  throws  on  the  difficult  subject  of  complementoids 
and  anticomplements.  We  have  seen  that  heated  serum  has  no 
complementary  activity,  but  that  its  injection  into  suitable  animals 
appears  to  call  forth  the  presence  of  anticomplements.  A  little 
consideration  will  show  that  this  apparent  anticomplementary 
action  can  be  explained  equally  well  by  the  absorption  of  the 
complements  in  a  specific  precipitate.  We  will  take  a  particular 
case. 

Goat  serum  heated  to  56°  C.,  and  therefore  containing  no  active 


BACTERIOLYSIS   AND   ALLIED    PHENOMENA 

complement,  is  injected  into  a  rabbit.  This  is  supposed  to  develop 
an  anticomplement  in  virtue  of  the  complementoids  it  contains ; 
we  know  that  it  also  develops  a  precipitin  which  combines 
with  the  proteids  of'  goat  serum,  and  in  doing  so  entangles  any 
complement  which  may  be  present.  Now  the  complement  of 
goat  serum  can  dissolve  ox  corpuscles  when  sensitized  by  a 
suitable  amboceptor  (e.g.,  serum  of  a  rabbit  which  has  been 
injected  with  ox  corpuscles).  When,  however,  the  serum  of  the 
normal  goat  is  mixed  with  that  of  the  rabbit  which  has  been 

<=V<7-txT"~ 

injected  with  rabbit  serum  and  used  in  this  way,  no  haemolysis 
occurs.  This  Ehrlich  explains  on  the  supposition  that  the  rabbit 
serum  contains  anticomplement,  but  it  is  also  explicable,  as 
Moreschi  and  Gay  have  shown,  on  the  supposition  that  the  com- 
plements are  all  absorbed  in  the  precipitate  (perhaps  an  invisible 
one)  formed  in  the  mixture. 

It  is  obvious  that  either  interpretation  may  be  the  correct  one, 
or  that  both  processes  may  come  into  play.  Moreschi  has 
investigated  the  process  further,  and  his  results  tend  to  show  that 
the  experiment  is  best  explained  on  the  absorption  of  complement 
theory,  and  not  by  the  presence  of  anticomplement.  One  of  his 
experiments,  and  that  very  ingenious,  will  be  described.  He 
injected  a  rabbit  with  hen's  egg  albumin,  and  obtained  a  precipitin 
to  that  substance ;  this  he  found  to  act  as  an  anticomplement 
to  fowl  serum,  but  not  to  that  of  the  rabbit,  guinea-pig,  or  goat, 
to  which,  of  course,  it  was  not  a  precipitin.  He  found,  however, 
that  if  he  added  to  any  of  these  latter  sera  a  minute  trace  (^30 ws 
of  its  volume)  of  egg-albumin,  it  appeared  to  become  so,  the 
explanation  being  that  a  precipitate  was  formed,  and  that  the 
complements  were  entangled  in  it. 

The  phenomenon  has  also  been  invoked  to  explain  some  extremely 
interesting  researches  of  PfeifTer  and  Friedberger,  who  thought 
they  had  demonstrated  the  presence  of  antibacteriolytic  substances 
in  normal  serum.  They  prepared  a  mixture  of  cholera  vibrios 
and  normal  serum,  which  they  allowed  to  stand  for  some  time,  and 
then  removed  the  bacteria  by  centrifugalization.  This  would 
remove  any  amboceptor  which  the  serum  might  contain,  and  they 
thought  that  they  could  demonstrate  that  an  anti-amboceptor 
still  remained.  They  added  to  the  serum  thus  prepared  some 
anticholera  serum  in  suitable  amount  and  a  lethal  dose  of  cholera 
vibrios,  and  injected  the  whole  into  an  animal,  which  invariably 
died ;  the  amount  of  antiserum  was  sufficient  to  protect  it  if  no 


172  THE    BORDET-GENGOU    PHENOMENON 

serum  which  had  been  exhausted  by  the  action  of  cholera  vibrio8 
had  been  added.  The  anti-amboceptor  which  thus  appeared  to 
be  present  in  normal  serum  seemed  to  be  strictly  specific  ;  a 
serum  which  had  been  weakened  by  the  action  of  cholera  vibrios 
had  no  action  on  antityphoid  serum,  and  vice  versa. 

Besredka  attempted  to  explain  these  findings  on  the  assumption 
that  "  free  receptors  "  passed  from  the  bacteria  into  the  normal 
serum,  and,  when  subsequently  mixed  with  antiserum,  combined 
with  it,  and  so  prevented  its  union  with  the  living  bacteria.  They 
showed,  in  reply,  that  normal  saline  solution  had  no  such  action ; 
but  this  is  hardly  conclusive,  since  receptors  are  dissolved  from 
the  bacteria  much  less  powerfully  in  normal  saline  than  in  serum. 

According  to  Sachs,  who  demonstrated  a  similar  occurrence  in 
haemolysis,  the  inhibiting  substance  which  actually  occurs  in  the 
serum  is  an  anticomplement.  Gay,  however,  believes  that  the 
whole  series  of  phenomena  can  be  explained  by  the  absorption  of 
the  complements  in  a  specific  precipitate.  Thus,  in  Sach's 
experiment  normal  rabbit  serum  heated  to  55°  C.  was  treated  with 
sheep's  corpuscles.  It  was  then  removed,  and  added  to  a  mixture 
of  heated  serum  immune  to  sheep's  corpuscles  (obtained  from  a 
rabbit  treated  with  sheep's  corpuscles),  fresh  guinea-pig's  serum, 
and  sheep's  corpuscles.  No  haemolysis  occurred,  as  it  did  in  the 
control  experiment,  in  which  no  "  exhausted  "  normal  serum  had 
been  added.  His  explanation  was  that  the  normal  serum  con- 
tained amboceptors  which  combined  with  sheep's  corpuscles, 
leaving  other  amboceptors  which  have  a  greater  affinity  for 
complement  than  those  combined  with  the  corpuscles  used  as  a 
test.  Gay's  explanation  (which  is  almost  certainly  the  correct 
one)  is  quite  similar  to  Moreschi's  explanation  of  the  anti- 
complements.  He  believes  that  the  immune  serum  contains  also 
a  precipitin  to  sheep's  serum,  and  that  in  the  "  exhaustion  "  of  the 
heated  normal  serum  by  the  sheep's  corpuscles  some  sheep's 
serum  was  added  to  it,  the  corpuscles  in  Sach's  experiment  not 
having  been  thoroughly  washed.  Thus  a  serum  precipitate  was 
formed,  and  the  alexins  or  complements  combined  therewith,  so 
that  no  haemolysis  occurred  on  the  subsequent  addition  of  sensitized 
corpuscles.  He  showed  that  if  thoroughly  washed  corpuscles 
were  used  for  the  exhaustion  the  phenomenon  did  not  occur,  and 
gives  other  experimental  proof. 

Gay  has  also  attempted  to  explain  the  process  of  deviation  of 
the  complements  in  bacteriolysis  (the  Neisser-Wechsberg  pheno- 


BACTERIOLYSIS    AND    ALLIED    PHENOMENA  173 

menon)  in  a  similar  way.  The  process  is  described  at  length  on 
a  subsequent  page,  but  consists  essentially  in  this :  when  bacteria 
are  acted  on  by  an  excess  of  amboceptor,  no  bacteriolysis  may 
occur,  although  sufficient  complement  is  present.  Gay's  researches 
on  this  point  have  not  been  fully  published  at  the  time  of  writing, 
but  he  apparently  thinks  that  soluble  portions — receptors — may 
pass  off  from  the  bacteria  into  the  fluid  in  which  the  latter  are 
suspended,  form  a  precipitate  with  the  serum,  and  absorb  the 
complement.  Much  evidence  will  be  required  before  this  can 
be  established ;  it  might  perhaps  explain  the  phenomenon  in 
vitro,  where  the  available  amount  of  complement  is  limited,  but 
the  effect  is  also  demonstrable  in  the  peritoneum,  where  we  should 
expect  as  much  complement  to  be  forthcoming  as  is  required. 

There  is  no  doubt  that  the  discovery  of  the  absorption  of  the 
complements  in  serum  precipitates  has  rendered  uncertain  many 
of  the  deductions  in  the  process  of  haemolysis  which  have  emanated 
from  the  Ehrlich  school,  and  the  whole  series  of  phenomena 
requires  re-investigation  in  the  light  of  the  observations  of 
Moreschi  and  Gay. 

Experiments  with  bacteriolytic  sera  soon  showed  that,  though 
they  protected  against  specific  infections,  they  only  did  so  in 
certain  doses.  It  is,  of  course,  readily  understandable  that  too 
small  an  amount  might  be  without  action,  but  it  was  also  found 
that  too  large  a  dose  might  be  equally  inefficacious.  Thus,  Loffler 
and  Abel  obtained  a  serum  which  protected  against  B.  coli,  and 
found  that  when  a  lethal  dose  of  this  organism  was  given  the 
animal  was  only  protected  when  it  had  received  between  0-02  and 
0*25  c.c.  of  serum,  larger  as  well  as  smaller  doses  being  equally 
without  effect.  Similar  results  have  been  observed  with  sera 
against  cholera,  dysentery,  malignant  oedema,  and  other  organisms, 
and  seem  to  be  general  in  dealing  with  bacteriolytic  sera  as 
opposed  to  antitoxin. 

They  can  also  be  obtained  in  vitro,  and  since  this  was  first 
shown  by  Neisser  and  Wechsberg,  the  phenomenon  is  often  called 
after  them.  Their  method  was  as  follows :  They  worked  with 
several  organisms,  amongst  others  with  V.  Afetchnikovi,  and  with 
the  serum  of  a  rabbit  which  had  been  immunized  against  this 
organism,  and  had  acquired  strong  bactericidal  powers.  Equal 
amounts  of  a  broth  culture  of  the  vibrio  were  placed  in  a  series 
of  test-tubes,  and  to  each  a  dose  of  heated  immune  serum  was 
added.  In  all  cases  a  uniform  amount  of  normal  rabbit  serum 


174 


THE    NEISSER-WECHSBERG    PHENOMENON 


(complement)  was  added,  and  the  mixture  made  up  to  constant 
volume  with  sterile  normal  saline  solution.  It  was  then  incubated 
for  three  hours  at  37°  C.,  and  finally  5  drops  from  each  tube 
were  plated  out  on  agar  and  incubated.  The  result  is  shown  on 
the  table. 


Amount  of  Broth 
Culture. 

Heated  Immune 
Serum. 

Fresh  Normal 
Serum. 

Colonies  Developing. 

I  C.C. 

Infinity. 

O'5  C.C. 

Infinity. 

0-25  c.c. 

Many  thousands. 

O'l  C.C. 

Several  hundreds. 

o'O5  c.c. 

About  100. 

rTHHJ  c-c-  in  aU 

0-025  c-c- 

0-3  c.c.  in  all 

About  50. 

cases. 

o*oi  c.c.                    cases. 

None. 

0-005  c.c. 

None. 

0-0025  c.c. 

About  100. 

O'OOI  C.C. 

Infinity. 

o'ooo5  c.c. 

Infinity. 

This  shows  that  when  g-^Vzr  c.c.  of  a  one-day-old  broth  culture  was 
treated  with  3  c.c.  of  fresh  normal  serum  (complement)  and  in- 
creasing doses  of  immune  serum,  there  was  no  appreciable  bacteri- 
cidal action  when  more  than  0-25  c.c.  of  the  latter  was  used,  or 
with  less  than  o-ooi  c.c.  When  the  amount  was  between  o-i  and 
0-025  c.c.,  or  about  0-0025  c.c.,  there  was  appreciable  action,  and 
when  it  was  between  o-oi  and  0-005  c.c.  it  was  complete. 

The  same  authors  also  demonstrated  a  very  remarkable  phe- 
nomenon of  exactly  the  same  nature.  A  normal  serum  which  has 
a  certain  amount  of  bactericidal  action  (owing  to  the  presence  of 
complement  and  of  a  small  quantity  of  amboceptor)  may  have  its 
action  increased,  diminished,  or  nullified  by  the  addition  of  a 
powerful  immune  serum.  This  is  shown  by  the  following  table, 
which  demonstrates  the  action  of  normal  guinea-pig  serum  on 
V.  Nordhafen  by  itself  and  after  the  addition  of  variable  doses 
of  heated  immune  serum. 

This  table  shows  that  there  is  a  definite  relation  between  the 
amounts  of  amboceptor  and  of  complement  which  have  to  be 
present  to  produce  a  maximal  effect.  In  the  case  of  the  normal 
serum,  it  is  evident  that  there  is  more  complement  than  can  be 
used  by  the  amount  of  amboceptor  present,  and  that  the  bacteri- 
cidal action  of  the  mixture  increases  as  immune  serum  is  added. 
0-05  c.c.  of  normal  serum  was  without  obvious  bactericidal  effect 
on  c.c.  of  the  culture,  whereas  the  same  amount  plus  0*01  c.c.  of 


BACTERIOLYSIS   AND    ALLIED    PHENOMENA 


175 


heated  immune  serum  destroyed  a  very  large  number  of  the 
bacteria.  When,  however,  the  immune  serum  is  added  in  larger 
proportion  than  this  it  does  harm  instead  of  good:  0-5  c.c.  of 


Number  of  Colonies  after  addition  of  Variable  Amounts  of 

Amount  of 

Amount  of 

Heated  Immune  Serum. 

Normal 

Culture. 

Serum. 

No  Serum 
added. 

i  c.c.  added. 

o'i  c.c.  added. 

0*01  c.c.  added. 

r-b-  c.c. 

I  C.C. 

None. 

Many 

A  few. 

None. 

in  all 

thousands. 

cases. 

0-5  c.c. 

None. 

Almost    in- 

About 100. 

None. 

finity. 

0-25  c.c. 

A  few. 

Infinity. 

Several 

A  few. 

_ 

hundreds. 

O'l  C.C. 

Several 

Infinity. 

Infinity. 

About  loo. 

thousands. 

0-05  c.c. 

Infinity. 

Infinity. 

Infinity. 

Many  hun- 

dreds. 

o-o25  c.c.    Infinity. 

Infinity. 

Infinity. 

Infinity. 

fresh  serum  completely  sterilizes  the  amount  of  the  culture  used, 
whereas  after  the  addition  of  i  c.c.  of  immune  serum  its  action 
is  barely  noticeable.  An  excess  of  amboceptor  shields  the  bacteria 
from  the  solvent  action  of  the  complements.  These  test-tube 
experiments  explain  the  results  obtained  in  vivo,  and  enable  us  to 
form  some  idea  as  to  the  mechanism  of  the  process. 

Neisser  and  Wechsberg  offered  the  following  explanation :  They 
assume  that  the  molecules  of  complement  and  amboceptor  unite 
in  the  mixture  before  the  latter  has  attached  itself  to  the  bacteria. 
Owing  to  the  excess  of  amboceptor,  there  will  not  be  sufficient 
molecules  of  complement  to  go  round,  and  it  will  follow  that  a 
variable  proportion  of  molecules  of  amboceptor  will  be  uncom- 
plemented. Unless  we  suppose  that  the  union  of  the  complement 
has  changed  the  affinity  of  the  cytophile  group  of  the  amboceptor 
for  the  bacterium,  it  will  follow  that  not  all  the  amboceptors  which 
attach  themselves  to  the  bacteria  will  be  charged  with  comple- 
ment, and  therefore  able  to  exert  a  bacteriolytic  or  solvent  action. 
To  take  a  concrete  case,  let  us  suppose  that  there  are  twice  as 
many  molecules  of  amboceptor  as  of  complement.  Half  the 
molecules  of  the  former  will  be  complemented,  and  it  will  follow 
that,  though  all  the  (appropriate)  receptors  of  the  bacterium  are 
occupied  by  amboceptor,  only  half  of  these  will  have  their  comple- 
mentophile  groups  occupied,  and  this  may  not  be  enough  to 
injure  it. 


176  DEVIATIONS    OF    COMPLEMENT 

They  also  imagine  the  possibility  of  the  combination  altering 
the  affinity  of  the  cytophile  group  of  the  amboceptor  for  the  cell. 
It  may  diminish  it,  in  which  case  fewer  complemented  molecules 


FIG.  42. — FIRST  POSSIBILITY. 

The  affinity  of  the  amboceptor  for  complement  is  unaltered,  as  a  result  of 
the  union  of  the  former  with  a  bacterium. 

of  amboceptor  would  attack  the  bacterium  than  ever,  and  the 
phenomenon  of  deviation  of  the  complements  would  be  even  more 
marked.  The  union  might  also  (conceivably)  increase  this  affinity ; 
in  this  case  the  amboceptors  which  were  complemented  would 


FIG.  43. — SECOND  POSSIBILITY. 

The  affinity  of  the  amboceptor  for  complement  is  diminished,  as  a  result  of  the 
union  of  the  former  with  a  bacterium. 

seize  on  the  bacterial  receptors,  to  the  exclusion  of  those  which 
were  not,  and  the  phenomenon  of  deviation  of  the  complements 
would  not  occur.  This  Neisser  and  Wechsberg  think  might  occur 
in  the  case  of  haemolysis,  for  in  this  process  deviation  has  not 


FIG.  44.— THIRD  POSSIBILITY. 
The  affinity  of  the  amboceptor  for  complement  is  increased. 

been  demonstrated,  and  solution  occurs  even  when  there  is  a 
great  excess  of  amboceptor.  In  the  case  of  haemolysis  by  snake 
venom,  however,  Myers  and  Stephens  showed  that  the  process 
might  only  take  place  when  medium  doses  of  venom  were  added, 


BACTERIOLYSIS   AND   ALLIED    PHENOMENA  177 

amounts  too  large  or  too  small  being  without  effect.  This  was 
also  corroborated  by  Kyes  and  Sachs,  who  showed  that  in 
presence  of  an  excess  of  venom  (amboceptor)  the  addition  of  a 
haemolyzing  amount  of  complement  might  be  without  effect. 
Now  Kyes  had  already  shown  that  venom  does  unite  directly  with 
one  of  the  substances  (lecithin)  which  can  complement  it,  and 
this  appeared  strong  evidence  in  favour  of  Neisser  and  Wechs- 
berg's  explanation.  But  Noguchi  afterwards  showed  that  the 
protective  action  of  large  doses  of  venom  protects  the  red  cor- 
puscles against  the  action  of  haemolytic  agents  other  than  comple- 
ments. Corpuscles  so  treated  are  not  dissolved  by  distilled  water 
or  by  tetanolysin.  It  appears,  therefore,  that  strong  solutions  of 
venom  have  a  curious  hardening  effect  on  red  corpuscles,  render- 
ing them  resistant  to  haemolytic  agents  of  all  sorts.  This  effect 
is  apparently  entirely  different  from  the  protection  of  a  bacterium 
by  excessive  doses  of  amboceptor,  and  should  not  be  used  as 
evidence  to  support  Neisser  and  Wechsberg's  explanation. 

Morgenroth  has  attempted  to  explain  the  absence  of  the  devia- 
tion of  the  complements  in  haemolysis  by  alleging  that  in  this  case 
the  amboceptors  and  complements  do  not  unite  until  the  former 
have  united  with  the  red  corpuscles ;  this,  of  course,  is  the  point 
at  issue.  He  finds  that  by  adding  anti-amboceptor  (for  the  cyto- 
phile  group)  to  a  mixture  of  amboceptor  and  complement  the 
phenomenon  of  deviation  can  be  reproduced.  But  his  experiment 
can  probably  be  explained  by  means  of  Gay's  observations,  to  be 
explained  subsequently,  and  in  any  ease  his  anti-amboceptor  would 
only  act  as  a  free  receptor  of  a  red  corpuscle,  and  the  amboceptor 
attached  to  it  would  be  in  the  same  condition  as  one  anchored  to 
a  corpuscle,  which  we  know  can  absorb  complement.  In  any  case, 
his  experiment  proves  little  or  npthing. 

Some  experiments  by  Meakins  afford  the  best  evidence  in 
favour  of  the  alteration  in  combining  affinity  undergone  by  ambo- 
ceptor after  union  with  complement,  and  call  for  short  reference, 
though  their  interpretation  is  somewhat  uncertain.  Meakins 
experimented  with  corpuscles  which  had  been  very  thoroughly 
washed  so  as  to  remove  all  trace  of  serum.  The  importance  of 
this  precaution  will  appear  subsequently.  He  found  that  when 
he  added  a  large  dose  of  heated  immune  serum  and  a  small  dose 
of  normal  serum  (complement)  to  corpuscles  thus  washed,  and 
allowed  them  to  act,  no  haemolysis  occurred.  The  corpuscles, 
however,  were  sensitized ;  for  when  they  were  centrifuged  down, 

12 


178     NEISSER-WECHSBERG    PHENOMENON    IN    HAEMOLYSIS 

washed,  and  treated  with  more  normal  serum,  haemolysis  took 
place.  It  thus  appears  that,  in  the  first  instance,  the  corpuscles 
took  up  only  those  amboceptors  which  had  no  attached  comple- 
ment. It  would  appear,  therefore,  that  amboceptor  may  unite 
with  complement  whilst  free,  and  that  when  it  has  done  so  its 
affinity  for  the  corpuscle  is  diminished.  This  is  one  of  the 
conditions  which  Neisser  and  Wechsberg  imagined,  and  which 
they  showed  would  make  the  phenomenon  of  deviation  even  more 
marked.  And  Meakins  shows,  though  not  in  a  very  clear  way, 
that  it  does  actually  take  place  with  corpuscles  that  are  well 
washed.  The  reason  why  it  does  not  take  place  under  ordinary 
circumstances  is  explicable  on  the  basis  of  Gengou's  reaction. 

But  this  theory  cannot  be  regarded  as  entirely  satisfactory.  It 
assumes,  in  the  first  place,  a  direct  union  between  complement 
and  amboceptor  before  the  latter  has  united  with  the  cell  or 
bacterium,  and  of  this  there  is  no  definite  evidence  apart  from 
this  phenomenon.  Ehrlich,  it  is  true,  supposes  a  feeble  union 
between  the  two  which  easily  dissociates,  even  at  low  temperatures, 
so  that  a  cell  which  has  been  placed  in  contact  with  a  mixture  of 
the  two  at  o°  will  become  saturated  with  amboceptor  devoid  of 
complement.  But  this  appears  to  be  a  fatal  objection  to  Neisser 
and  Wechsberg's  explanation  ;  for  if  the  hypothetical  amboceptor- 
complement  combination  dissociates  readily  at  a  low  temperature, 
it  should  do  so  still  more  at  a  high  one.  Now  the  receptor- 
amboceptor-complement  will  not  dissociate,  or  only  to  a  very 
slight  extent,  since  lytic  action  will  at  once  commence.  It  follows 
that  the  amboceptor-complement  molecules  will  gradually  all 
dissociate,  and  the  complements  will  sooner  or  later  find  their 
way  to  the  anchored  amboceptors,  and  there  remain.  The  process 
may  be  delayed,  but  after  a  time  all  the  attached  amboceptors 
will  become  complemented.  And  if,  as  seems  implied  in  Ehrlich's 
writings,  the  affinity  of  the  complementophile  group  becomes 
raised  in  virtue  of  the  union  of  the  cytophile  group,  with  its 
appropriate  receptor,  this  process  will  take  place  all  the  more 
quickly. 

It  appears  much  more  likely  that  the  Neisser- Wechsberg 
phenomenon  is  an  example  of  a  reaction  of  a  kind  which  has 
already  attracted  much  attention,  and  which  may  possibly  modify 
fundamentally  our  views  on  the  antibodies,  toxins,  etc.  They  will 
be  referred  to  again  when  we  deal  with  the  agglutinins,  and  it  is 
sufficient  to  say  here  that  in  other  cases,  when  there  is  no  question 


BACTERIOLYSIS   AND    ALLIED    PHENOMENA  179 

of  two  bodies,  and  therefore  of  the  validity  of  Neisser  and 
Wechsberg's  explanation,  antibodies  and  other  substances  have 
their  action  diminished  or  suspended  when  they  are  present  in 
excess ;  thus  with  a  great  excess  of  agglutinin  there  may  be  no 
agglutination.  And  Detre  and  Sellei  have  shown  that  phenomena 
which  are  apparently  somewhat  similar  occur  in  the  haemolysis 
induced  by  mercury  perchloride. 

In  view  of  the  important  role  in  immunity  which  must  be 
ascribed  to  alexin — and,  as  we  shall  see,  it  acts  not  only  as  a 
bacteriolytic  agent,  but  also  plays  a  part  of  great  importance  in 
phagocytosis — an  inquiry  into  its  source  becomes  of  paramount 
importance.  The  theory  was  put  forward  very  early  in  the 
history  of  the  subject,  before  the  mode  of  action  of  alexin  and  its 
dependence  on  immune  body  were  understood,  that  it  was  derived 
from  the  leucocytes,  either  by  a  process  of  secretion  or  disintegra- 
tion. This  was  first  suggested  by  Hankin,  who  prepared  a 
substance  having  bacteriolytic  powers  from  extracts  of  the 
lymphatic  glands  and  spleen ;  this,  however,  was  probably  not  a 
true  alexin.  Experimental  evidence  of  a  more  convincing  kind 
soon  followed ;  thus  Denys,  Van  de  Velde,  and  others,  caused  the 
production  of  aseptic  exudates  rich  in  leucocytes  by  injecting 
various  irritants  into  the  pleural  cavity  of  animals,  and  found  that 
the  more  leucocytes  present  the  greater  the  bactericidal  action  of 
the  fluid.  Buchner  performed  similar  experiments,  using  aleuron 
emulsions  to  produce  the  exudate,  and  found  that  the  richly 
cellular  material  had  a  greater  bactericidal  effect  than  blood  or 
serum ;  in  his  experiments  he  avoided  the  possibility  of  phago- 
cytosis (which  probably  explained  some  of  the  results  of  other 
investigators)  by  freezing  the  fluids  before  determining  their 
potency.  A  large  number  of  other  researches  were  made  about 
this  time,  and  all  pointed  in  the  same  direction,  and  in  the  early 
nineties  it  was  fairly  generally  held  that  the  alexins  were  given  off 
in  some  way  or  another  by  the  leucocytes.  This  view  was  strongly 
held  by  Metchnikoff,  whose  microcytase  must  be  regarded  as 
identical  with  the  alexin  or  complement  which  acts  on  the 
bacteria;  and  this  microcytase  he  holds  to  be  produced  by  the 
polynuclear  leucocytes  under  certain  circumstances,  which  will  be 
discussed  subsequently. 

With  the  discovery  of  the  compound  nature  of  the  bactericidal 
substances  the  question  entered  on  a  new  phase,  and  new  methods 
of  investigation  were  seen  to  be  necessary.  Since  then  very 

12 — 2 


l8o  ORIGIN    OF    COMPLEMENT 

many  researches  have  appeared,  the  point  at  issue  being  whether 
the  alexin  or  complement  is  formed  from  the  polynuclear  leuco- 
cytes (for  there  is  no  evidence  pointing  to  the  lymphocytes). 
Some  of  the  most  important  of  these  must  be  briefly  summarized. 

1.  Various   observers  have   investigated  the  relation  between 
the  number  of   leucocytes  in  a  given  fluid  and  the  amount  of 
complementary    action.      Thus    Bulloch,    working   with    hsemo- 
complement,  found  its  amount  closely  proportionate  to  the  number 
of  polynuclear  leucocytes.     His  figures  are  very  convincing,  and 
the  only  alternative  explanation  of  his  results  is  that  the  injection 
of  any  substance  which  stimulates  leucocytosis  stimulates  at  the 
same  time  the  hypothetical  complement-producing  organ  or  tissue. 
Similar  observations  have  been  made  under  natural  conditions 
by  Longcope,  who  found  that  in  disease  in  man  the  occurrence 
of  hyperleucocytosis  is  accompanied  by  a  rise  in  the  amount  of 
complement.      On    the    other   hand,    Guseff's   investigations   on 
similar  lines  were  entirely  negative,  and  he  was  unable  to  trace 
any  parallelism   between   the   two ;    and    Briscoe,   dealing   with 
peritoneal  fluids,  arrived  at  a  similar  result.     These  results  may 
be  taken  as  typical  of  the  whole.     A  series  of  experiments  by  an 
able  worker  is  apparently  conclusive  in  one  direction,  and  appears 
to  establish  the  point  beyond  controversy,  but  is  immediately  met 
with  another  on  similar  lines,  pointing  to  a  diametrically  opposite 
result. 

Other  investigations  on  similar  lines  are  those  of  Bordet,  who 
found  that  the  oedema  fluid  poured  out  in  passive  congestion  in 
an  immunized  animal — a  fluid  poor  in  cells — contains  immune 
body,  but  no  complement,  and  that  the  aqueous  humour  is  entirely 
devoid  of  both  substances.  This  latter  fact  has  been  corroborated 
by  Levaditi,  who  adds  that  when,  after  tapping,  the  aqueous 
humour  becomes  rich  in  leucocytes,  it  acquires  alexin  at  the  same 
time. 

2.  Many   observers    have   attempted    to   prepare   complement 
from   emulsions   of   polynuclear   leucocytes,  from   bone-marrow, 
or  from  lymphoid  organs.     The  results  have  been  diverse  in  the 
extreme,  and  this  diversity  appears  due  in  part  to  the  fact  that 
substances  other  than   true  alexins  or  complement  may  cause, 
or    play   some   part   in   the   causation   of,   both   haemolysis   and 
bacteriolysis.     In  particular,  we  may  refer  to  the  important  role 
of  lecithin  and  its  allies  in  haemolysis,  and  possibly  in  the  destruc- 
tion of  bacteria  also.     It  is  quite  possible,  for  instance,  that  the 


BACTERIOLYSIS    AND    ALLIED    PHENOMENA  l8l 

haemolytic  complement  prepared  by  Levaditi  from  autolyzed 
lymphoid  tissue  was  one  of  these  lipoid  substances ;  it  was 
soluble  in  alcohol  and  thermostable.  The  substance  prepared  by 
Morgenroth  and  Korschun  from  extracts  of  various  organs  was 
probably  similar  in  nature,  since  they  showed  it  to  be  thermostable, 
and  not  to  give  rise  to  the  production  of  antibodies  on  injection. 
It  may  be  at  once  admitted,  as  pointed  out  by  Conradi,  that 
autolytic  changes  occurring  in  various  organs  give  rise  to  the 
production  of  bactericidal  substances ;  these  may,  perhaps,  be 
organic  acids  or  other  simple  substances,  but  they  do  not  help  us 
in  the  inquiry  as  to  whether  complement  (having  the  power  to 
dissolve  sensitized  bacteria  or  red  corpuscles)  is  formed  from 
the  leucocytes. 

The  most  important  researches  on  this  point  are  those  of 
Schattenfroh  and  Petrie,  and  they  are  mutually  contradictory. 
Schattenfroh  washed  the  leucocytes  thoroughly  in  saline  solution, 
added  it  to  the  heated  serum,  and  obtained  a  bactericidal  effect. 
His  results  were  corroborated  by  Lastchenko.  Petrie's  researches 
were  carried  out  with  great  care,  and  he  was  especially  particular 
to  remove  every  trace  of  complement  adhering  to  the  leucocytes 
by  repeated  washing  in  normal  saline  solution.  (Ascher  had 
previously  shown  that  some  trace  might  remain  when  the  process 
had  only  been  carried  out  three  times.)  The  leucocytes  were 
obtained  from  aleuron  exudates,  and  after  washing  they  were 
frozen  to  the  temperature  of  liquid  air,  and  ground  to  an  im- 
palpable powder,  the  method  used  being  that  of  Macfadyen  for 
the  preparation  of  endotoxin.  His  results  were  entirely  negative, 
and  he  failed  entirely  to  reactivate  heated  bactericidal  serum  by 
any  of  his  extracts.  Lambotte  failed  equally,  working  on  lines 
more  like  those  of  Schattenfroh. 

Petrie's  experiments  seem  conclusive  on  the  point  which  he 
investigated,  and  it  seems  quite  certain  that  polynuclear  leucocytes 
do  not  contain  alexin  or  complement  as  such  ;  they  do  not,  how- 
ever, negative  the  possibility  that  they  may  contain  a  precursor  of 
this  substance,  which  may  either  be  secreted  or  set  free  during 
the  natural  solution  of  the  leucocyte,  in  either  case  being  converted 
into  an  active  form  during  the  process.  Both  these  views  have 
been  widely  held,  and  we  shall  discuss  them  later. 

3.  In  the  process  of  coagulation  of  the  blood  large  numbers  of 
the  leucocytes,  and  especially  the  polynuclears,  are  disintegrated 
and  partially  dissolved,  and  these  disintegrated  leucocytes  are 


l82  ORIGIN    OF    COMPLEMENT 

generally  accepted  as  the  source  of  the  fibrin  ferment.  If  the 
complements  are  also  set  free  during  the  solution  of  the  leucocytes, 
there  should  be  far  more  present  in  the  serum  than  in  the  circu- 
lating plasma ;  it  might  happen,  indeed,  that  the  plasma  might  be 
devoid  of  any  complementary  action,  though  this  does  not  neces- 
sarily follow. 

The  difficulty  of  investigating  this  subject  is  very  great,  and 
the  evidence  is  mostly  indirect.  As  far  as  it  goes,  it  seems  to 
point  to  the  fact  that  the  plasma  is  alexin-free,  or,  at  any  rate, 
poorer  than  the  serum.  The  main  direct  researches  into  this 
subject  are  those  of  Gengou  and  Falloise,  and  here,  again,  they 
point  in  a  diametrically  opposite  direction.  Gengou  (whose  results 
have  been  accepted  in  their  entirety  by  Metchnikoff)  worked  with 
plasma  obtained  by  preventing  coagulation  by  collecting  the  blood 
in  paraffined  tubes  and  centrifugalizing  forthwith — a  method  cer- 
tainly less  open  to  objections  than  any  dependent  on  the  addition 
of  anticoagulants,  but  difficult  in  execution.  He  found  that  plasma 
thus  prepared  was  devoid  of  bactericidal  action,  though  the  serum 
from  the  same  animal  possessed  it.  In  some  cases  the  plasma 
betrayed  some  action,  perhaps  because  a  partial  coagulation  had 
taken  place,  but  it  was  always  less  potent  than  the  serum.  These 
results  have  been  opposed  by  numerous  investigators,  using  various 
methods  of  inhibiting  coagulation.  Thus,  Falloise  used  intra- 
venous injections  of  peptone,  the  addition  of  sodium  oxalate  or 
fluoride  ;  Petterson  used  oxalate  and  citrate,  and  Hahn  histon, 
and  in  all  cases  no  appreciable  difference  in  alexin  content  between 
the  plasma  and  serum  was  found.  But  this  is  hardly  a  fair  test. 
Peptone  is  generally  believed  to  be  a  leucotoxic  or  leucolytic 
agent ;  and  in  the  case  of  the  citrated  or  oxalated  blood  it  is 
obvious  that  (if  leucocytes  are  the  origin  of  fibrin  ferment)  some 
leucolysis  has  occurred,  since  the  cell-free  fluid  coagulates  on  the 
addition  of  calcium.  Falloise  also  worked  with  paraffin  tubes  on 
Gengou's  method,  and  found  that  the  plasma  contained  as  much 
haemolytic  complement  as  the  serum.  Lastly,  both  he  and 
Lambotte  worked  with  plasma  obtained  by  centrifugalizing  (or 
by  allowing  sedimentation  to  occur  in)  the  blood  in  a  vein  isolated 
between  two  ligatures  and  kept  at  a  low  temperature,  and  with 
the  same  results.  Delezenne,  however,  criticizes  these  results  by 
pointing  out  that  plasma  obtained  in  this  way  coagulates  instantly 
in  glass  tubes  at  the  ordinary  temperature,  a  phenomenon  in- 
dicating that  fibrin  ferment  is  present,  and  that  leucocytes  have 


BACTERIOLYSIS    AND    ALLIED    PHENOMENA  183 

become  disintegrated,  or  have  at  least  performed  an  act  of  secretion 
which  they  do  not  perform  in  the  living  blood.  The  conditions, 
therefore,  are  not  altogether  natural. 

We  are  forced  to  rely  to  a  large  extent  on  indirect  evidence,  and 
this,  as  far  as  it  goes,  is  strongly  in  favour  of  Gengou's  view. 

4.  Ainley  Walker's  experiments  dealt  with  the  amount  of  com- 
plement present  in  the  successive  amounts  of  serum  squeezed  out 
from  a  clot,  and  showed  that  this  gradually  rises,  the  first  few  drops 
containing  but  little,  and  the  quantity  gradually  increasing  until  it 
attains  its  maximum  in  about  twenty-four  hours.  Other  explana- 
tions are  possible,  but  it  is  at  least  probable  that  the  cause  of  this 
increase  is  the  destruction  of  the  leucocytes  which  is  known  to 
occur  during  the  process  of  coagulation,  and  that  the  formation  of 
complement  is  quite  analogous  with  that  of  fibrin  ferment.  The 
serum  first  formed  is  weak  in  complement,  the  destruction  of  the 
leucocytes  having  only  just  commenced ;  and  it  seems  a  fair 
deduction  that,  if  we  could  examine  the  plasma  when  no  leucolysis 
has  occurred,  we  should  find  that  it  contains  much  less,  or  none 
at  all. 

This  view  is  supported  by  Levaditi  in  a  series  of  researches  on 
the  fate  of  cholera  vibrios  injected  into  the  circulatory  system  of 
immunized  guinea-pigs,  care  being  taken  to  avoid  as  far  as  possible 
the  destruction  of  leucocytes.  Under  these  circumstances  he 
finds  that  the  Pfeiffer  phenomenon  does  not  take  place,  or  does  so 
much  more  slowly ;  the  vibrios  circulate  in  the  blood  for  half  an 
hour  or  more  without  showing  the  least  trace  of  granular  trans- 
formation, and  are  rapidly  taken  up  by  the  leucocytes.  In  a 
similar  series  of  experiments  on  haemolysis  he  was  able  to  prove 
the  existence  of  sensitized  red  corpuscles  in  the  circulation, 
although  the  animal  did  not  present  the  least  trace  of  haemo- 
globinuria,  and  was  of  opinion  that  this  was  due  to  the  absence  of 
free  alexin  in  the  plasrrr.  The  anaemia  due  to  the  injection  of 
haemolytic  amboceptor  he  believes  to  be  brought  about  by  the 
destruction  of  the  red  corpuscles  within  the  macrophages  of  the 
spleen. 

As  a  third  item  of  indirect  evidence  we  may  quote  the  experi- 
ments of  Lubarsch  and  others,  to  the  effect  that  an  animal  may  be 
killed  by  the  intravenous  injection  of  a  smaller  number  of  bacteria 
(e.g.,  of  anthrax)  than  are  destroyed  by  a  small  quantity  (i  c.c.  or 
less)  of  serum.  The  simplest  explanation  is  that  the  blood  contains 
an  abundance  of  immune  body,  and  the  failure  of  the  defensive 


184  ORIGIN    OF    COMPLEMENT   AND    IMMUNE    BODY 

mechanism  is  due  to  lack  of  complement.  It  would,  however,  be 
unwise  to  attach  too  much  importance  to  this  proof. 

The  difficulties  in  coming  to  a  conclusion  on  this  subject  are 
great,  but  on  the  whole  it  would  appear  that  the  evidence  is  some- 
what in  favour  of  the  views  of  Metchnikoff,  and  that  in  all  proba- 
bility there  is  no  free  alexin,  or  but  little,  in  the  plasma,  and  that 
the  main  source  of  this  substance  is  the  polynuclear  leucocyte, 
but  we  cannot  regard  this  as  definitely  proved.  This  being  so, 
the  discussion  as  to  whether  the  formation  of  alexin  is  a  vital 
secretory  process  or  a  phenomenon  of  cell  death  and  solution 
cannot  be  regarded  as  of  great  importance.  Here  again  the 
experimental  work  is  most  inconclusive,  Lastchenko  holding  that 
living  leucocytes  give  off  alexin  when  suspended  in  heated  serum, 
whilst  Lazar  found  that  it  is  only  set  free  when  some  of  the 
leucocytes  are  destroyed.  Kanthack's  experiments  may  perhaps 
be  quoted  at  this  point.  He  found  that  anthrax  bacilli  immersed 
in  frog's  lymph  become  surrounded  by  eosinophile  cells.  After  a 
time  these  cells  discharge  their  granules,  and  the  bacilli  soon 
begin  to  show  signs  of  injury,  becoming  less  refractile  and  losing 
their  sharp-cut  outline.  After  this  these  leucocytes  move  away 
from  the  bacilli,  from  which  we  might  argue  that  they  are  un- 
injured, and  that  the  solution  of  their  granules  is  a  vital  action, 
and  exactly  equivalent  to  the  secretion  of  pepsin  by  the  gastric 
cells.  This,  of  course,  does  not  exclude  the  possibility  that  alexin 
might  also  be  liberated  during  the  process  of  phagolysis,  as 
Metchnikoff  maintains. 

As  regards  the  origin  of  the  immune  body  the  evidence  is  unani- 
mous, showing  that  it  originates  from  the  lymphoid  tissues,  and 
probably  from  the  mononuclear  leucocytes.  It  is  found  that  if 
animals  be  injected  with  cholera  vibrios,  killed  at  varying  intervals 
afterwards,  and  extracts  made  of  the  different  organs,  the  first  sites 
in  which  the  antibodies  make  their  appearance  are  the  lymphoid 
organs,  especially  the  bone-marrow,  spleen,  and  lymphatic  glands. 
Similar  facts  have  been  observed  for  other  bacteria  and  for  red 
blood-corpuscles  (Pfeiffer  and  Marx,  Deutsch,  Wassermann).  And 
in  Bulloch's  experiments  the  amount  of  haemolytic  amboceptor  was 
found  to  run  roughly  parallel  with  the  number  of  mononuclear 
leucocytes  present  in  the  blood.  In  all  probability  this  increase  of 
circulating  mononuclears  must  be  regarded  in  such  cases  as  an 
indication  of  the  activity  of  the  lymphoid  organs,  which  are  to  be 
regarded  as  the  main  source  of  the  protective  antibodies.  This  is 
a  generalization  of  the  highest  importance  in  immunity,  and  it  is 


BACTERIOLYSIS    AND    ALLIED    PHENOMENA  185 

fortunate  that  the  evidence  in  its  favour  is  clear  and  direct ;  and  it 
may  be  added  that  the  proof  is  strengthened  by  the  demonstration 
of  the  fact  that  the  agglutinins  are  formed  in  the  same  region.  It 
seems  clear  that  the  main,  if  not  the  only,  function  of  the  lympho- 
cyte is  the  elaboration  of  the  defensive  antibodies. 

There  is  no  doubt  that  immune  body,  like  the  other  antibodies, 
circulates  as  such  in  the  plasma.  The  most  striking  proof  is  that 
of  Metchnikoff  in  regard  to  spermotoxin,  but  other  evidence  is 
available,  and  the  point  is  not  disputed. 

Methods  of  Researches  on  Immune  Bodies  and 
Complements. 

The  methods  employed  in  the  investigation  of  the  haemolytic 
sera  are  fairly  simple  in  theory,  though,  as  a  rule,  somewhat 
tedious  in  practice,  owing  to  the  necessity,  in  most  cases,  for 
quantitative  work  (so  that  slight  degrees  of  haemolysis  may  not  be 
overlooked),  and  for  numerous  controls.  The  animal  used  in  the 
preparation  of  the  immune  serum  will  naturally  depend  on  the 
corpuscles  to  be  employed,  but  in  most  cases  the  rabbit  is  most 
convenient.  The  corpuscles  used  for  immunization  should  be 
collected  in  normal  saline  solution  containing  about  i  per  cent, 
sodium  citrate,  centrifugalized,  and  rewashed  three  or  four  times 
in  normal  saline  solution,  so  as  to  remove  every  trace  of  serum, 
complements,  etc.  If  this  is  not  done  the  resulting  serum  may  be 
of  great  complexity,  and  results  obtained  by  its  action  misleading. 
The  emulsion  used  for  the  injection  may  contain  about  half  its 
volume  of  corpuscles,  and  some  10  c.c.  (or  more  in  the  case  of 
large  rabbits)  may  be  injected  into  the  peritoneum.  In  most 
cases  three  such  injections  at  weekly  intervals  will  cause  the  pro- 
duction of  a  powerful  haemolytic  serum,  but  if  there  is  any  doubt 
5  c.c.  or  so  of  blood  may  be  withdrawn  from  the  marginal  vein  of 
the  ear,  and  used  to  test  the  progress  of  the  immunization.  The 
ear  is  well  rubbed  to  make  it  hyperaemic,  shaved,  and  washed  with 
alcohol.  A  small  puncture  is  now  made  with  a  flat  surgical 
needle  or  fine  knife,  taking  care  that  the  vessel  is  merely  incised 
and  not  completely  divided.  As  a  rule  the  blood  will  now  flow 
quickly,  drop  by  drop,  and  a  sufficient  amount  may  be  collected  in 
a  sterile  test-tube.  If  the  flow  is  sluggish  the  blood  may  be 
milked  out  by  passing  the  finger  gently  along  the  vein. 

When  the  immunization  is  complete  the  rabbit  is  usually  killed, 
and  as  much  blood  as  possible  is  collected.  The  best  way  to  do  this 


l86  HAEMOLYSIS — METHODS    OF    RESEARCH 

is  to  administer  chloroform,  and  insert  a  cannula  into  the  carotid 
artery ;  an  easier  method  is  to  stick  a  small  piece  of  capillary 
glass  tubing  through  the  wall  of  the  heart  whilst  it  is  still  beating, 
the  animal  being,  of  course,  under  chloroform.  In  either  case  the 
blood  is  led  into  a  sterile  tube,  allowed  to  clot — preferably  in  the 
incubator,  as  the  retraction  of  the  clot  is  thereby  increased — and 
the  clear  serum  'withdrawn  by  means  of  a  sterile  pipette,  and 
stored  in  sterile  tubes  or  small  flasks,  which  may  be  plugged  with 
cotton- wool  or  hermetically  sealed.  Where  the  serum  is  to  be  heated 
to  destroy  complement  this  may  be  accomplished  in  a  thermostat, 
or  more  simply  by  sealing  it  in  a  narrow  tube  which  is  tied  to  the 
bulb  of  a  thermometer,  and  placed  in  a  beaker  of  warm  water,  and 
the  temperature  regulated  by  hand  by  the  application  and  removal 
of  a  spirit-lamp.  By  this  means  any  desired  temperature  can  be 
maintained  within  very  close  limits,  and  the  necessity  for  main- 
taining several  thermostats  or  of  altering  the  regulator  is 
removed. 

The  emulsion  of  corpuscles  to  be  employed  in  the  actual  testing 
is  prepared  as  above,  and  must  be  rewashed  at  least  four  times, 
since  it  is  found  that  traces  of  complement  may  remain  after  three 
washings.  The  emulsion  is  usually  made  up  so  that  it  contains 
5  per  cent,  of  corpuscles.  This  is  readily  accomplished  by  per- 
forming the  last  centrifugalization  in  a  graduated  tube,  going  on 
until  the  volume  is  constant.  The  bulk  of  the  corpuscles  is  then 
read  off,  and,  the  necessary  amount  of  normal  saline  solution 
being  added,  the  corpuscles  thoroughly  mixed  in.  It  is  advisable 
that  aseptic  precautions  should  be  observed  throughout,  but  as 
this  is  troublesome  it  can  often  be  omitted.  It  must  be  remem- 
bered, however,  that  many  common  bacteria  produce  haemolysis, 
and  that  if  the  mixtures  of  corpuscles,  serum,  etc.,  be  incubated 
for  long  periods  fallacies  may  arise  from  this  cause. 

The  actual  experiment  in  its  simplest  form  is  carried  out  as 
follows  :  The  necessary  amounts  of  5  per  cent,  emulsion  of 
corpuscles,  heated  serum,  and  complement  are  placed  in  a  narrow 
test-tube,  and  in  most  cases  normal  saline  solution  is  added  to 
bring  the  whole  up  to  a  definite  volume.  This  is  now  incubated 
for  two  hours,  being  stirred  or  shaken  once  or  twice  in  the  mean- 
time. It  is  now  removed  and  placed  in  a  vertical  position  in  the 
ice-chest  for  twelve  hours  or  so  and  examined.  If  there  is  com- 
plete haemolysis  the  fluid  will  be  deeply  coloured,  and  there  will  be 
no  sediment,  or  only  a  minute  deposit  of  stromata.  With  partial 
haemolysis  the  fluid  will  be  less  deeply-coloured,  and  there  will  be 


BACTERIOLYSIS    AND   ALLIED    PHENOMENA 


i87 


a  deposit  of  undissolved  corpuscles,  and  when  there  is  no  haemo- 
lysis the  fluid  will  be  untinged. 

Modifications  of  the  process  are  numerous,  and  almost  every 
investigator  has  his  own.  Thus  it  is  very  convenient  to  make  the 
mixtures  by  means  of  one  of  Wright's  pipettes.  The  whole  is 
sucked  into  the  pipette,  which  is  sealed  and  incubated.  At  the 
end  of  the  process  the  mixture  may  be  expelled  on  to  white  filter- 
paper.  Any  unaltered  corpuscles  will  form  a  solid  dark  deposit  in 
the  centre  of  the  drop,  whilst  the  fluid  which  soaks  through  the 
paper  will  be  tinged  or  colourless,  according  to  the  amount  of 
haemolysis  which  has  taken  place.  The  amount  of  haemolysis  may 
also  be  determined  more  accurately  by  comparison  of  the  super- 
natant fluid  with  a  series  of  colour-standards  previously  prepared. 
The  mere  presence  or  absence  of  the  phenomenon  may  be  readily 
shown  by  dropping  some  of  the  fluid  on  to  filter-paper. 

The  amount  of  immune  body  present  may  be  determined  by 
some  such  process  as  the  following  :  In  each  of  a  series  of  narrow 
test-tubes  (having  a  mark  to  indicate  2  c.c.)  is  placed  i  c.c.  of  the 
emulsion  of  corpuscles,  and  then  varying  amounts  of  the  heated  im- 
mune serum — e.g.,  o-ooi  c.c.,  0-0025  c.c.,  0-005  c-c->  etc->  or  more  if 
the  serum  be  a  weaker  one.  As  these  small  amounts  are  not  easy  to 
measure  accurately,  the  serum  may  be  diluted  ten  or  a  hundred 
times  with  normal  saline  solution  and  suitable  multiples,  these 
amounts  taken  in  the  case  of  the  smaller  doses.  The  actual 
measurements  are  done  with  graduated  pipettes,  which  can  be 
procured  from  any  instrument-maker.  The  complementing  serum 
is  then  added :  the  amount  necessary  to  dissolve  i  c.c.  of  fully- 
sensitized  serum  should  have  been  previously  determined  by  a 
few  rough  tests  (we  will  suppose  it  to  be  0-2  c.c.).  Lastly,  sufficient 
normal  saline  is  added  to  bring  the  volume  of  each  tube  up  to  2  c.c., 
and  the  whole  series  treated  as  above.  Thus — 


No. 

Emulsion  of 
Corpuscles. 

Heated  Immune 
Serum. 

Fresh 
Serum. 

Haemolysis. 

I. 

ICC. 

o-ooi  c.c. 

O'2  C.C. 

None. 

2. 

0-0025  c.c. 

M 

3- 

0-005  c.c. 

5  i 

4- 

0-0075  c.c. 

Trace. 

5- 

O'OI  C.C. 

Partial. 

6. 

0-025  c-c- 

Complete. 

7- 

0-015  c  c- 

„ 

8. 

0-0175  c.c. 

»> 

9- 

O'O2  C.C. 

f  , 

10. 

0-025  c'c- 

•• 

l88  BACTERIOLYSIS — METHODS    OF    RESEARCH 

Here  0-0125  c>c-  °f  ^he  immune  serum  contained  sufficient 
immune  body  to  sensitize  fully  i  c.c.  of  a  5  per  cent,  emulsion  of 
corpuscles — i.e.,  a  given  volume  of  serum  will  sensitize  1-25  of  its 
own  volume  of  corpuscles. 

The  determination  of  the  amount  of  complement  is  made  by  an 
inversion  of  this  method.  Thus  Gay,  who  has  made  numerous 
investigations  as  to  the  amount  of  complement  present  in  human 
serum,  proceeds  as  follows  :  The  sensitizing  serum  is  derived  from 
a  rabbit  which  has  been  injected  with  ox  corpuscles.  This  is 
heated,  and  the  amount  necessary  for  complete  sensitization  of  a 
definite  amount  of  ox  corpuscles  is  determined;  thus  in  his 
experiment  0-7  c.c.  saturated  7  c.c.  of  a  5  per  cent,  emulsion.  A 
series  of  tubes,  each  containing  i  c.c.  of  a  5  per  cent,  emulsion  of 
fully-sensitized  corpuscles,  is  prepared,  and  varying  doses  of  the 
serum  to  be  tested  are  added  ;  the  amount,  which  is  small,  is  pre- 
pared by  dilution  with  normal  saline  to  such  an  extent  that  the 
actual  bulk  added  is  0*1  c.c.  The  subsequent  treatment  is  as 
above.  Gay  and  Ayer  find  that  on  the  average  about  -£$  c.c.  has 
to  be  added  to  bring  about  complete  haemolysis,  the  limits  being 
i1^  and  g^  c.c. 

Quantitative  researches  on  the  bacteriolytic  action  of  the  serum 
are  very  much  more  difficult.  The  actual  determination  of  the 
amount  of  bactericidal  action  is  by  no  means  easy,  and  the  results 
obtained  are  of  very  little  importance,  since  the  serum  may  be 
very  deficient  in  complement,  and  deviation  may  occur.  The 
method  which  has  been  chiefly  employed  is  that  of  plating  out 
after  the  bacteria  and  serum  have  been  allowed  to  act  together  at 
incubator  temperature  for  a  given  period.  The  method  is  briefly 
as  follows  :  The  emulsion  of  bacteria  must  be  of  constant  strength. 
As  a  rule,  it  is  sufficient  to  take  a  twenty-four-hour  broth  culture, 
and  to  dilute  it  to  the  same  degree  in  all  experiments ;  or  the 
same  loop  may  be  employed  throughout,  or  some  one  or  other  of 
the  counting  methods  which  have  been  described  may  be  used. 
The  emulsions  should  be  dilute,  so  that  all  the  bacteria  may 
be  killed.  -  Klien  recommends  i  :  8,000  of  a  twenty-four-hour 
broth  culture  in  the  case  of  B.  typhosus.  Lastly,  normal  saline 
solution  is  better  than  broth  as  a  diluting  agent,  since  it  diminishes 
the  chance  of  error  owing  to  the  multiplication  of  bacteria  during 
the  somewhat  lengthy  process  of  preparing  the  dilutions. 

The  actual  process  is  as  follows  :  Measured  small  amounts  of 
the  serum  to  be  tested  are  placed  in  a  series  of  tubes,  a  uniform 


BACTERIOLYSIS    AND    ALLIED    PHENOMENA  189 

amount  of  the  emulsion  added,  each  tube  made  up  to  a  definite 
volume,  and  all  incubated  for  one  to  four  hours.  At  the  end  of 
this  period  a  uniform  quantity  is  withdrawn  from  each,  and 
plates  prepared  either  by  mixing  with  melted  agar,  or  gelatin 
where  suitable,  or  by  smearing  over  ready-poured  agar  plates. 
The  amount  must,  of  course,  be  the  same  in  each  case,  and  may 
be  easily  withdrawn  by  means  of  one  of  Wright's  pipettes,  which 
is  sterilized  after  use  by  being  washed  out  several  times  with 
boiling  water  or  oil  at  150°  C.  The  plates  are  then  incubated,  and 
the  colonies  which  develop  after  twenty-four  or  forty-eight  hours 
are  enumerated,  and  the  amount  of  serum  which  kills  all  or  the 
greatest  number  of  bacteria  is  noted. 

Certain  controls  are  necessary,  the  main  being — (a)  a  tube 
inoculated  as  above,  but  without  the  addition  of  serum ;  and 
(b)  a  tube  also  containing  bacterial  emulsion,  and  also  a  relatively 
large  amount  of  heated  serum.  The  main  error  comes  in  from 
the  reduction  of  the  number  of  colonies  in  consequence  of  aggluti- 
nation, but  this  can  be  discounted  in  some  measure  by  comparison 
with  the  plate  prepared  from  control  (&). 

Other  methods  are  employed,  notably  that  of  Wright,  for  which 
the  original  article  should  be  consulted.  The  value  of  the  pro- 
cesses is  not  great,  since  it  does  not  tell  us  even  the  actual 
bactericidal  value  of  the  circulating  blood  (since  we  do  not  know 
the  amount  of  complement  which  is  available)  nor  the  amount  of 
immune  body.  In  some  cases  a  serum  containing  a  large  amount 
of  the  latter  substance  will  show  little  or  no  bactericidal  power  in 
vitro,  owing  to  the  deficiency  in  complement,  and  may  require  the 
addition  of  a  hundred  times  its  volume  of  normal  serum  to  be 
fully  complemented. 

To  determine  the  relative  amount  of  immune  body  present,  the 
principle  of  the  method  used  for  the  measurement  of  the  haemolytic 
amboceptor  is  adopted,  a  series  of  mixtures  of  constant  amounts 
of  bacterial  emulsion  and  fresh  normal  serum  is  prepared,  and 
varying  amounts  of  the  heated  immune  serum  to  be  tested  are 
added,  the  whole  made  up  to  uniform  volume,  and  treated  as 
above.  Here  a  further  control  is  necessary,  since  the  fresh 
normal  serum  may  contain  some  immune  body  or  be  otherwise 
bactericidal.  One  of  Neisser  and  Wechsberg's  examples  of  this 
process  has  been  already  quoted. 

The  determination  of  the  amount  of  bactericidal  complement  is 
simple  enough  theoretically,  and  follows  the  same  lines  as  that 


IQO  THE    CYTOLYSINS 

for  the  determination  of  the  haemolytic  complement.  In  actual 
practice  these  procedures  are  all  so  tedious  that  most  of  the 
measurements  of  complement  have  been  made  on  the  latter 
variety ;  the  two  are  believed  to  have  the  same  origin,  and  there 
is  no  reason  to  think  that  the  one  does  not  run  parallel  to  the 
other.  Gay  and  Ayer  employ  a  more  direct  method,  adding 
varying  amounts  of  the  serum  to  be  tested  to  a  definite  volume 
(0*5  c.c.)  of  a  suspension  of  cholera  vibrios,  prepared  by  emulsify- 
ing four  twenty-four-hour  agar  cultures  in  10  c.c.  of  normal  saline, 
and  subsequently  adding  a  sufficient  sensitizing  dose  of  serum 
from  an  immunized  rabbit.  The  action  is  allowed  to  go  on  for 
one  and  a  half  hours  at  37°  C.,  films  prepared,  stained,  and 
examined  as  to  the  degree  of  the  changes  undergone  by  the 
vibrios.  They  found  that  ^-^  c.c.  of  normal  human  serum  was 
sufficient  to  cause  a  complete  Pfeiffer's  reaction  in  0-5  c.c.  of 
cholera  emulsion  tested  as  above,  whilst  when  y^^  c.c.  was  used 
there  were  distinct  changes. 

The  Cytolysins. 

Bordet's  discovery  of  acquired  haemolytic  powers,  arising  from 
the  injection  of  foreign  red  corpuscles,  proved  the  starting-point 
of  a  most  interesting  series  of  researches,  for  it  was  soon  shown 
that  the  phenomenon  was  not  an  isolated  one,  but  that  it  might  be 
produced  when  almost  any  animal  cell  took  the  place  of  the  red 
corpuscles.  Thus,  Metchnikoff  in  1899  prepared  a  leucotoxic  serum 
by  the  injection  of  the  cells  from  the  spleen  of  a  rat  (mostly 
lymphocytes)  into  a  guinea-pig.  The  serum  of  the  latter  agglu- 
tinated and  partially  dissolved  the  leucocytes,  the  lymphocytes 
being  most  affected.  Besredka  studied  the  subject,  and  found 
that,  as  in  the  hgemolysins,  two  substances — one  thermostable 
(sensibilatrice  or  amboceptor)  and  one  thermolabile  (alexin  or 
complement) — took  part  in  the  reaction.  He  studied  the  speci- 
ficity of  the  substance,  and  found  it  was  not  sharply  specialized 
in  its  action  to  leucocytes  of  the  animal  used  for  the  source  of  the 
antigen  ;  it  would  attack  those  of  most  animals,  but  not  man.  It 
was  toxic,  3  c.c.  of  serum  being  a  lethal  dose.  He  also  prepared 
an  antileucotoxin. 

The  next  cytolysin  to  be  prepared  (by  Landsteiner,  and  inde- 
pendently by  Metchnikoff)  was  spermotoxin.  This  was  a  very 
suitable  subject  for  study,  since  its  action  could  be  readily 


BACTERIOLYSIS    AND    ALLIED    PHENOMENA  IQI 

observed,  the  cells  on  which  it  acted  being  motile  ;  and  it  must  be 
pointed  out  that  these  cytolysins  do  not  cause  complete  solution  of 
the  cells.  A  red  blood-corpuscle  is  a  remarkable  object,  and 
macroscopic  evidence  of  its  (partial)  solution  is  easily  obtained. 
It  is  otherwise  with  the  cytolysins,  and  here  refined  histological 
methods  are  often  necessary  for  the  demonstration  of  a  solvent 
action.  Agglutination  of  a  suitable  suspension  of  the  cells  is, 
however,  invariably  present,  and  is  easily  observed.  Further 
evidence  is  also  obtainable  by  observing  the  action  of  the  serum 
on  live  animals  and  the  disturbances  in  function  which  it  produces. 
In  the  case  of  the  spermotoxin,  the  spermatozoa  are  rendered 
immotile,  and  are  agglutinated,  but  are  not  dissolved. 

Several  interesting  phenomena  were  brought  to  light  by  a  study 
of  spermotoxin.  Thus,  Moxter  showed  that  its  action  is.  not 
sharply  specific,  since  a  spermotoxic  serum  is  also  haemolytic. 
MetchnikofT  thought  that  this  non-specificity  is  only  apparent, 
since  haemolytic  sera  are  not  spermotoxic ;  and  he  succeeded  in 
removing  the  haemolytic  substance  from  the  serum  by  the  addition 
of  red  corpuscles,  leaving  the  spermotoxin  intact. 

It  may  be  pointed  out  here  that  similar  results  have  been 
obtained  with  the  other  cytolytic  sera ;  they  are  not  sharply 
specific,  all  being  haemolytic,  and  some  attacking  several  cells,  as 
well  as  those  which  have  been  used  as  their  antigens.  This 
subject  has  been  thoroughly  investigated  by  Pearce.  Some  of 
his  results  may  be  briefly  epitomized.  Haemolytic  sera  act,  of 
course,  most  strongly  on  the  red  corpuscles,  which  they  lake,  and 
give  rise  to  haemoglobinuria.  They  also  produce  fatty  degeneration 
of  the  renal  epithelium  and  necrosis  of  the  cells  of  the  liver. 
With  very  small  doses  there  may  be  no  haemoglobinuria,  bile- 
pigment  being  present  in  the  urine,  but  the  lesions  of  the  liver 
and  kidney  are  also  present.  A  serum  prepared  by  the  injection 
of  kidney  cells,  thoroughly  washed,  so  that  no  blood  was  injected 
with  them,  was  haemolytic  in  vitro,  but  did  not  produce  haemo- 
globinuria. It  caused  albuminuria,  with  presence  of  casts  and 
granular  degeneration  of  the  liver  cells.  A  serum  similarly  pre- 
pared from  the  suprarenal  glands  had  no  action  on  them,  but 
produced  granular  or  fatty  degeneration  of  the  kidney  and  liver. 
An  animal  injected  therewith  showed  immediate  pallor  of  the 
mucous  membranes  and  cardiac  and  respiratory  failure.  He 
found  that  hepatotoxins  and  pancreatotoxins  were  without  specific 
action,  behaving  simply  like  haemolysins. 


TRICHOTOXIN,    HEPATOTOXIN,    NEPHROTOXIN 

It  is  obvious  that  these  results  are  readily  explicable  if  we 
assume  that  the  red  corpuscles  and  tissue  cells  have  receptors  in 
common,  but  that  a  particular  sort  of  receptor  is  most  abundant  in 
a  particular  species  of  cell.  But,  according  to  Beebe,  sera  which 
are  much  more  sharply  specific  can  be  prepared  if,  instead  of 
injecting  the  cells  themselves,  we  employ  the  nucleo-proteid  pre- 
pared from  them  ;  the  method  had  also  been  employed  by  Bierry 
and  Pettit  in  the  case  of  the  nucleo-proteids  of  the  liver  and 
kidney. 

Another  serum  which  was  prepared  early  in  the  history  of  the 
subject  was  trichotoxin,  the  cytotoxin  for  the  ciliated  epithelium. 
This  also,  as  Von  Dungern  showed,  had  a  haemolytic  action, 
though  he  considered  that  there  were  no  red  corpuscles  in  the 
substance  used  for  the  injections. 

Hepatotoxin  is  produced  by  the  injection  of  emulsions  of  liver 
cells  or  of  nucleo-proteid  prepared  from  the  liver.  It  causes  con- 
gestion of  the  liver,  fatty  or  granular  degeneration  of  the  proto- 
plasm, and  dilatation  of  the  bile  canaliculi.  If  the  serum  has 
been  prepared  by  means  of  nucleo-proteid,  no  other  organ  is 
affected.  But  the  effects  of  hepatotoxin  may  also  be  produced  by 
nephrotoxic  and  lienotoxic  serum,  etc. 

A  considerable  amount  of  interesting  work  has  been  done  on 
nephrotoxin,  and  the  questions  which  have  arisen  are  far  from 
having  been  settled.  It  is  produced  in  the  usual  way,  by  injection 
of  animals  with  a  fine  emulsion  of  kidney  cells  (well  washed 
to  remove  blood-corpuscles,  etc.)  from  a  foreign  species.  It 
produces  albuminuria  (but  no  glycosuria,  according  to  Bierry), 
and  symptoms  having  at  least  some  resemblance  to  uraemia  (coma, 
etc.)  are  occasionally  produced.  These  symptoms  are  not  specific, 
and  are  frequently  caused  by  injections  of  other  cytolysins  (SlpHpo- 
toxin,  etc.),  or  of  emulsions  of  foreign  cells.  We  have  already 
pointed  out  that  Beebe  and  others  have  claimed  to  be  able  to 
produce  a  truly  specific  nephrotoxin  by  means  of  injections  of 
nucleo-proteid  from  the  kidney. 

Of  more  interest  is  the  question  of  the  possible  formation  of 
an  autonephrotoxic  body,  which  might  conceivably  be  produced 
when  part  of  a  kidney  becomes  disorganized  whilst  in  the  living 
body.  It  has  been  thought,  for  instance,  that  when  a  toxin  acts 
on  the  kidneys  it  produces  death  and  subsequent  solution  of  the 
renal  epithelium,  and  that  these  soluble  substances,  being  absorbed 
into  the  system,  call  forth  an  autonephrotoxin,  which  reacts  on 


BACTERIOLYSIS   AND   ALLIED    PHENOMENA 

the  kidney,  dissolving  more  cells,  which  produce  more  of  the  anti- 
body, a  vicious  circle  being  thus  produced.  Hence  a  pathology 
for  nephritis  and  uraemia  on  quite  new  lines  was  suggested  by 
Ascoli  and  Figari  and  Lindeman,  etc.  Thus  the  cardiac  hyper- 
trophy of  renal  disease  is  supposed  to  be  due  to  a  spasm  of  the 
peripheral  vessels  and  increase  of  blood-pressure  due  to  the 
nephrotoxic  serum ;  the  nervous  symptoms  on  the  supposition 
that  there  is  a  neurotoxin  produced  concurrently  with  the  nephro- 
toxin,  and  spontaneous  recovery  by  the  production  of  an  anti-auto- 
nephrotoxin,  a  substance  for  the  existence  of  which  there  is  a 
little  evidence. 

There  is  a  certain  amount  of  experimental  confirmation  of  this 
theory.  Thus  Lindeman  treated  dogs  with  potassium  bichromate, 
causing  nephritis,  and  found  that  the  serum  of  these  animals 
(though  free  from  bichromate)  was  toxic  for  other  dogs.  Again, 
Le  Play  and  Corpechot  found  that  the  injection  of  renal  tissue 
(of  the  guinea-pig)  into  the  rabbit  produced  important  organic 
lesions :  great  increase  in  volume,  fibrosis  of  the  connective 
tissues,  cystic  dilatations  of  the  tubules,  and  desquamation  of  the 
renal  epithelium.  That  these  changes  may  be  due  to  the  produc- 
tion of  a  nephrolysin  appears  possible  from  the  fact  that  when 
these  injections  are  made  in  gravid  animals  similar  appearances 
may  be  seen  in  the  kidneys  of  the  foatus,  suggesting  that  the 
nephrolysins  traverse  the  placenta  (Charrin  and  Delaware). 
Albarran  and  Bernard  also  found  that  renal  tissue  is  lethal  on 
injection,  but  Pearce  denies  this,  and  holds  that  their  animals 
were  killed  by  bacterial  infection.  Further,  Nefedieff  ligatured 
one  ureter  (in  the  rabbit),  and  found  changes  similar  to  those 
seen  in  chronic  nephritis.  His  results,  might,  of  course,  have 
been  due  to  the  formation  of  a  nephrotoxin  in  consequence  of 
the  disintegration  of  the  renal  cells  subsequent  to  ligature  of 
the  ureter ;  but  Albarran  pointed  out  that,  according  to  Nefedieff 
himself,  the  second  kidney  was  unaffected  at  a  time  when  the 
serum  was  nephrotoxic,  as  tested  on  other  animals.  Sheldon 
Amos  failed  to  reproduce  Nefedieff's  results;  according  to  her, 
ligature  of  one  ureter  causes  death  after  an  average  period  of  sixty- 
nine  and  a  half  days  in  the  guinea-pig,  and  fifty-two  days  in  the 
rabbit.  There  may  be  lesions  on  the  control  side,  but  if  so  these 
are  slight,  and  the  liver  is  also  affected.  But  that  these  results  are 
due  to  the  action  of  a  nephrotoxic  serum  appears  most  unlikely, 
from  the  fact  that  when  the  whole  pedicle  of  the  kidney,  or  the 


IQ4  GASTROTOXIN 

artery  and  vein,  are  ligatured,  no  such  results  follow,  though  the 
whole  substance  of  the  kidney  is  absorbed.  These  and  other 
researches  make  it  very  doubtful  whether  the  facts  observed  in 
nephritis  are  explicable  on  the  nephrotoxic  theory  alone,  but 
further  information  on  the  subject  is  needed. 

The  degree  of  specificity  of  the  nephrotoxic  serum  is  not  yet 
settled.  According  to  Pearce,  the  lesions  which  it  produces  may 
be  caused  by  other  sera.  This  has  been  confirmed  by  other 
observers,  but  Woltmann,  though  in  accordance  with  Pearce  on 
the  main  question,  thinks  that  nephrotoxin  does  exhibit  some 
degree  of  specificity :  it  produces  marked  congestion  of  the 
medulla  and  swelling  of  the  cortex,  results  not  seen  with  other 
sera.  Beebe  also  finds  nephrotoxic  sera  produced  by  the  injection 
of  nucleo-proteid  prepared  from  the  kidney  cause  renal  lesions, 
whereas  other  cytotoxic  sera  produced  by  the  injection  of  other 
nucleo-proteids  do  not. 

Gastvotoxic  serum  is  especially  interesting  in  view  of  its  possible 
action  in  the  production  of  gastric  ulcer.  It  has  been  very 
thoroughly  studied  by  Bolton,  and  was  prepared  by  injecting 
rabbits  with  emulsions  or  extracts  of  guinea-pig's  gastric  mucous 
membrane  into  the  rabbit.  The  serum  thus  obtained  was  injected 
into  guinea-pigs,  and  was  found  to  be  lethal,  even  in  small  doses 
(i  to  5  c.c.) ;  a  dose  of  10  c.c.  usually  caused  death  in  twenty-four 
hours.  The  lesions  were  confined  to  the  stomach,  and  were 
striking  and  characteristic.  They  consisted  of  patches  of  necrosis 
extending  down  to  the  muscularis  mucosae,  and  often  surrounded 
by  a  haemorrhagic  infiltration  of  the  surrounding  tissues.  After 
a  time  this  necrotic  tissue  disappeared,  leaving  an  ulcer  presenting 
some  resemblance  to  the  ordinary  acute  gastric  ulcer.  These 
appearances  (necrosis,  etc.)  were  not  seen  if  the  acidity  of  the 
gastric  juice  was  neutralized  by  alkalis.  No  very  definite  action 
could  be  demonstrated  on  gastric  mucous  membrane  in  vitro,  but 
isolated  cells  exposed  to  the  action  of  the  serum  became  hyaline 
in  appearance,  resembling  shadows.  Further,  the  serum  had  a 
powerfully  agglutinating  action  on  gastric  cells,  and  produced  a 
precipitate  in  clear  solutions  obtained  by  filtration  through  a 
Berkefeld  filter. 

Interesting  facts  were  discovered  as  regards  its  specificity.  It 
is  haemolytic,  but  this  appears  to  be  due  to  the  fact  that  it 
contains  haemolysin  as  well  as  gastrotoxin.  This  is  shown  as 
follows :  If  the  serum  is  heated  it  loses  its  power  to  produce  the 


BACTERIOLYSIS    AND   ALLIED    PHENOMENA 

characteristic  necrosis  of  the  stomach,  so  that  its  immune  body 
cannot  be  reactivated  by  guinea-pig  alexin;  but  the  latter  body 
can  reactivate  haemolysin  prepared  by  immunizing  rabbits  with 
guinea-pig's  corpuscles.  If  the  serum  is  placed  in  contact  with 
an  emulsion  of  guinea-pig's  mucous  membrane,  it  becomes 
innocuous,  both  immune  bodies  being  absorbed;  but  when 
saturated  with  red  corpuscles,  it  loses  its  haemolytic  power,  and 
retains  its  necrotizing  properties. 

Rabbits  injected  with  emulsions  of  rabbit's  mucous  membrane 
develop  a  gastrotoxin  which  acts  on  guinea-pigs,  but  not  on  the 
rabbit  itself.  Similarly  for  guinea-pigs  treated  with  emulsions  of 
mucous  membrane  from  the  same  species :  their  serum  becomes 
gastrotoxic  for  the  rabbit,  not  for  the  guinea-pig. 

To  account  for  these  remarkable  facts  it  is  suggested  that  the 
gastrotoxin  has  two  cytophile  groups — one  which  combines  with 
the  gastric  cells  of  the  animal  which  produces  it,  and  one  which 
combines  with  those  of  the  other  species.  Thus  the  gastrotoxin 
of  the  rabbit  has  a  cytophile  group,  a,  which  has  an  affinity  for 
rabbit's  gastric  cells,  and  a  second,  b,  which  unites  with  those  of 
the  guinea-pig.  During  the  process  of  immunization  the  animal 
produces  an  anti-immune  body,  which  combines  with  the  cytophile 
group  a,  but  not  with  b.  This  is  readily  explicable  on  the  side- 
chain  theory.  It  follows,  therefore,  that  the  gastrotoxin  is  never 
efficacious  against  the  species  which  produces  it,  being  always 
neutralized  as  regards  these  cells  by  a  partial  anti-antibody. 

A nti -intestinal  serum  has  been  prepared.  It  is  extremely  toxic, 
causing  gangrene  of  the  mucous  membrane  and  death.  Less 
powerful  sera  cause  non-fatal  diarrhoea. 

Syncytiolysin,  or  placentolysin,  has  been  obtained  by  injections  of 
emulsions  of  placental  tissue.  According  to  Liepman  the  serum 
thus  obtained  will  give  a  precipitate  with  a  solution  of  placental 
tissue,  with  blood  from  the  umbilical  vein,  or  even  with  that  of 
a  gravid  woman,  but  not  that  of  a  non-gravid  woman  or  a  man ; 
hence  he  proposed  a  serum  test  for  pregnancy.  But  his  results, 
which  seemed  highly  improbable,  have  been  disproved  by 
Weichardt,  who  showed  that  the  serum  thus  obtained  acts  equally 
well  on  placental  solutions  and  on  all  human  blood.  The  question 
of  the  action  of  the  placenta  when  injected  (in  a  fine  emulsion) 
into  the  tissues  is  of  some  importance  in  connection  with  a  possible 
pathology  for  eclampsia  and  the  nephritis  of  pregnancy.  Most 
authorities  (though  not  all)  find  that  the  animal  thus  treated 

13—2 


196  SYNCYTIOTOXIN,  NEUROTOXIN 

develops  nephritis  and  lesions  of  the  liver.  Now  it  is  known  that 
in  some  cases  at  least  fragments  of  the  placenta  break  loose  and 
circulate  for  a  time  in  the  blood  during  pregnancy,  and  it  is  not 
difficult  to  suppose  that  dissolved  products  of  these  cells  are 
constantly  being  absorbed.  Hence  it  seems  possible  that  some 
at  least  of  the  cases  of  nephritis  during  pregnancy  and  of  eclampsia 
may  be  produced  in  this  way ;  and  Weichardt  produced  symptoms 
resembling  those  of  eclampsia  by  macerating  placental  tissue  with 
syncytiolysin,  and  injecting  the  result  into  normal  rabbits.  Hence 
it  was  hoped  that  an  antitoxin  for  puerperal  eclampsia  and 
nephritis  might  be  produced  by  immunizing  animals  with  placental 
tissue,  so  as  to  produce  a  serum  which  would  dissolve  the  circulat- 
ing placental  cells,  and  prevent  the  destruction  of  the  cells  of  the 
liver  and  kidneys.  This  does  not  seem  to  have  been  put  into 
practice,  and  there  are  numerous  theoretical  objections  which 
might  be  raised. 

Prostatotoxin  has  been  prepared  by  Jungano  by  injecting  an 
emulsion  of  the  prostates  of  young  dogs  into  rabbits.  The  serum 
clumps  emulsions  of  prostatic  cells,  and  when  injected  in  vivo 
produces  fatty  and  granular  degeneration  of  the  epithelial  cells 
of  the  gland  and  a  leucocytic  infiltration  of  the  stroma;  it  is 
apparently  fairly  specific,  there  being  no  obvious  lesion  of  other 
organs. 

Neurotoxin  has  been  prepared  by  Delezenne,  Centanni,  Delille, 
and  others,  by  the  treatment  of  one  animal  with  the  brain  sub- 
stance from  another,  which  is  often  in  itself  somewhat  toxic,  so 
that  the  process  does  not  always  succeed.  It  causes  a  remarkable 
series  of  phenomena  indicative  of  profound  intoxication  of  the 
nerve  centres.  These  usually  begin  with  somnolence  and  torpor, 
which  come  on  shortly  after  the  injection,  and  may  last  some 
hours,  being  succeeded  by  convulsive  crises,  in  which  there  are 
tonic  and  clonic  spasms ;  there  may  be  one  such  attack,  or  a 
series,  with  coma  between  each.  The  temperature  is  lowered,  and 
death  usually  occurs  in  one  to  twenty- four  hours.  The  histological 
changes  are  marked,  and  affect  the  ganglion  and  cortical  cells; 
they  indicate  a  profound  degree  of  destruction  of  these  structures 
(neurolysis).  The  substance  is  most  active  when  injected  into 
the  brain  direct ;  when  introduced  into  the  veins  it  is  innocuous, 
but  forms  an  anticytolysin. 

Schmidt  has  prepared  a  serum  which  he  claims  to  be  more  or  less 
specific  for  the  peripheral  nerves.  A  guinea-pig  which  is  injected 


BACTERIOLYSIS    AND   ALLIED    PHENOMENA  IQ7 

with  an  emulsion  of  the  sciatic  nerves  of  frogs  develops  in  its 
serum  a  substance  which  leads,  when  injected  into  frogs,  to  the 
rapid  production  of  symptoms  of  paralysis,  which  may  become 
complete,  and  resemble  Landry's  paralysis  in  man.  Most  of  the 
animals  die  in  from  twelve  to  forty-eight  hours,  and  their  nerves 
show  fragmentation  of  the  axis  cylinders,  multiplication  of  the 
nuclei  in  the  sheath  of  Schwann,  etc.  The  serum  is  also 
haemolytic  for  frog's  corpuscles,  but  neither  normal  serum  nor  a 
simple  haemolytic  serum  produce  these  paralytic  symptoms. 

The  suggestion  has  been  made  that  sympathetic  ophthalmia 
might  be  due  to  a  specific  cytotoxin  formed  by  the  disintegration 
and  absorption  of  the  iris  and  ciliary  body  in  the  injured  eye 
(Bram  Pusey).  There  is  a  certain  amount  of  experimental  proof 
in  favour  of  this  interesting  theory.  Thus  Le  Play  and  Corpechot 
prepared  an  ophthalmotoxic  serum,  and  found  that  animals 
injected  therewith  were  less  resistant  than  normal  animals  to 
injections  of  B.pyocyaneus  into  the  anterior  chamber.  The  subject 
has  been  more  fully  investigated  by  Golovine,  who  prepared  his 
serum  by  injecting  into  rabbits  an  emulsion  of  the  ciliary  bodies 
of  the  dog  (twelve  to  twenty  in  each  animal).  The  ophthalmo- 
toxic serum  thus  obtained  was  tested  by  injection  into  the  anterior 
chamber.  It  led  to  the  production  of  a  slight  pericorneal  injection, 
a  fibrinous  exudate  into  the  anterior  chamber,  and  some  appear- 
ances of  iritis.  Microscopically  it  was  found  that  the  ciliary 
processes  presented  evidence  of  inflammation  and  degeneration, 
being  infiltrated  with  leucocytes  containing  granules  of  pigment. 
There  was  also  marked  evidence  of  degeneration  of  the  epithelium 
covering  these  processes.  When  the  serum  was  injected  into  the 
veins  the  macroscopic  effects  were  not  observed,  but  similar 
microscopic  changes  were  noted  in  the  epithelium. 

The  pigment  taken  up  by  the  leucocytes  was  derived  from  the 
ciliary  processes,  which  may  become  almost  absolutely  decolourized. 
Hence  Golovine  holds  that  his  serum  contains  not  only  a  specific 
cyclotoxin,  but  also  a  pigmentolysin. 

Other  cytolytic  sera  have  been  prepared,  but  are  not  of  much 
interest.  A  reference  may  be  made  to  thyrotoxic  serum,  which 
has  been  used  in  the  treatment  of  exophthalmic  goitre,  though 
without  any  considerable  success.  Indeed,  the  use  of  cytolytic 
sera  has  proved  most  disappointing  in  practice.  An  anti-epithelial 
serum  which  was  very  early  suggested  as  a  cure  for  cancer,  but 
proved  inefficacious,  and  others  have  been  tried.  There  are  very 


198  BACTERICIDAL    SERA 

many  problems  connected  with  cytolytic  action  that  are  unsolved, 
and  there  can  be  but  little  doubt  that  future  research  in  this 
direction  will  yield  results  of  great  pathological  importance,  both 
in  theory  and  in  practice,  and  whether  the  therapeutical  advance 
will  take  the  form  of  a  potent  serum  or  of  a  juster  knowledge  of 
the  inner  processes  of  the  body  in  disease  the  future  will  show. 

THERAPEUTIC  APPLICATIONS  OF  BACTERICIDAL  SERA. 

The  discovery  of  the  great  therapeutic  value  of  diphtheria  anti- 
toxin naturally  led  to  attempts  at  antitoxin  treatment  of  other 
diseases,  but  it  was  soon  found  that  it  was  impossible  to  prepare 
a  potent  toxin,  and  therefore  antitoxin,  in  the  great  majority  of 
cases.  The  discovery  of  Pfeiffer's  phenomenon,  and  the  sub- 
sequent researches  on  bacteriolysis  and  haemolysis,  with  the 
demonstration  of  the  nature  of  substances  at  work,  indicated  that 
the  problem  was  to  be  solved,  if  at  all,  on  other  lines,  and  anti- 
sera  were  made  by  injecting  the  bacteria  themselves  into  suitable 
animals.  The  process  need  not  be  described  at  length,  and  of 
course  slight  modifications  are  necessary  in  different  cases.  In 
general  the  early  part  of  the  treatment  consists  in  the  injection 
of  small  doses  of  dead  or  avirulent  bacteria,  or  in  some  cases 
(e.g.,  anthrax)  of  a  more  virulent  vaccine  and  of  a  protective  serum 
from  an  already  immunized  animal.  The  animal  (horses,  donkeys, 
or  goats,  are  usually  employed)  is  thus  immunized,  and  now  large 
doses  of  virulent  bacteria  are  given  in  order  to  stimulate  the 
production  of  antibodies  to  as  great  an  extent  as  possible.  This 
part  of  the  treatment  is  often  prolonged,  and  may  last  for  a  year 
or  more.  At  the  end  of  this  time  the  animal  is  bled  in  the 
manner  described  above,  and  the  serum  used  for  protective  or 
curative  purposes.  In  some  cases  it  is  standardized,  the  usual 
method  being  to  determine  the  amount  which  will  just  protect 
a  small  animal  from  a  lethal  dose  of  living  bacteria,  or  from  some 
multiple  thereof.  Thus  Sclavo's  serum  is  tested  by  injecting 
1*6  c.c.  into  six  rabbits,  each  of  which  receives  shortly  afterwards 
a  known  dose  of  virulent  bacilli ;  if  three  of  the  animals  survive, 
and  the  rest  have  their  lives  greatly  prolonged  (as  compared  with 
controls),  the  serum  is  considered  to  be  efficacious.  Antistrepto- 
coccic  serum  may  be  standardized  in  a  similar  way :  according  to 
Hewlett,  0-05  c.c.  should  suffice  to  preserve  a  rabbit  from  ten 
lethal  doses  of  living  streptococci  injected  intravenously.  In 
other  cases  a  somewhat  more  refined  method  is  adopted,  and  the 


BACTERIOLYSIS   AND    ALLIED    PHENOMENA  IQQ 

amount  of  antibody  present  is  estimated.  In  the  case  of  anti- 
typhoid serum  the  simplest  method  is  to  measure  the  degree  of 
agglutination,  which  may  rise  as  high  as  i  :  1,000,000.  This 
cannot  be  taken  as  an  absolute  criterion  of  the  amount  of  bactericidal 
substance  present,  but  in  the  great  majority  of  cases  the  two 
antibodies  are  developed  roughly  proportionately,  and  the  agglu- 
tination may  be  taken  as  a  fair  guide.  Of  course,  the  bacteriolytic 
potency  may  be  worked  out  by  the  method  already  described, 
taking  care  that  a  sufficient  amount  of  complement  is  added,  and 
that  there  is  no  deviation.  This  is  probably  the  best  method,  and 
is  sometimes  employed;  thus  Shiga  found  that  O'oooi  c.c.  of  his 
antidysentery  serum  when  reactivated  by  0-3  c.c.  of  fresh  serum 
would  kill  all  the  bacilli  in  ^^  milligramme  of  a  one-day-old  agar 
culture. 

The  results  of  tests  of  this  nature  have  been  to  show  that 
extremely  potent  sera  can  be  obtained  against  typhoid  bacilli, 
cholera  vibrios,  dysentery  bacilli,  and  perhaps  streptococci ;  sera 
of  less  but  still  of  some  power  against  plague  bacilli,  anthrax 
bacilli,  pneumococci,  the  gonococcus,  and  the  meningococcus ; 
whilst  the  results  with  staphylococci  and  tubercle  bacilli  have 
been  to  all  intents  and  purposes  negative. 

The  method  of  action  of  these  sera  is  not  quite  settled.  In 
some  cases  there  is  an  abundance  of  bactericidal  immune  body, 
and  there  is  no  reason  to  doubt  that,  when  employed  as  a  prophy- 
lactic agent,  this  becomes  complemented  in  the  animal  body,  and 
causes  bacteriolysis  of  the  infecting  organism.  This  is  certainly 
the  case  with  the  sera  directed  against  typhoid  fever,  cholera,  and 
dysentery.  In  other  sera,  which  are,  nevertheless,  of  definite 
protective  and  even  curative  value,  this  effect  cannot  be  demon- 
strated. This  is  the  case  with  anti-anthrax  serum.  Here  we 
have  to  assume  either  that  the  substance  owes  its  value  to  the 
presence  of  opsonins  or  of  anti-endotoxin,  or  possibly  (in  some 
cases)  that  it  may  contain  free  toxins,  or  at  least  specific  antigens, 
and  act  as  a  vaccine,  producing  active  rather  than  passive 
immunity,  as  was  suggested  by  Wright  in  the  case  of  Calmette's 
typhoid  serum,  which  is  prepared  in  a  manner  somewhat  different 
from  that  just  described.  There  is  some  reason  for  thinking  that 
Sclavo's  serum  acts  opsonically,  and  with  regard  to  the  presence 
of  anti-endotoxin,  it  may  be  pointed  out  that  the  prolonged  course  of 
immunization  usually  employed  may  lead  to  the  production  of 
this  substance  in  small  amounts. 


200  CLINICAL    FAILURE    OF    BACTERICIDAL    SERA 

These  sera,  which  for  the  purpose  of  convenience  we  shall 
consider  together  as  if  they  were  all  bactericidal,  are  in  general 
protective,  but  not  curative.  Thus  the  clinical  use  of  antityphoid 
and  anticholera  serum  has  shown  them  to  be  quite  worthless  or 
even  dangerous ;  dysentery  serum  is  of  distinct  value  if  used 
early  in  the  attack,  and  some  of  the  other  sera  are  of  some  value, 
and  their  use  is  discussed  in  the  final  section  of  this  book.  In 
general  terms,  however,  and  comparing  them  with  diphtheria 
antitoxin,  we  may  say  that  they  have  proved  most  disappointing 
in  practice.  The  reason  for  this  failure  requires  some  discussion. 

Antibacterial  sera  as  ordinarily  used  are,  of  course,  devoid  of 
complement,  which  has  usually  disappeared  long  before  use ;  it 
is  rendered  inert  on  keeping,  and  is  especially  susceptible  to  the 
antiseptics  commonly  added  as  a  preservative.  The  first  sugges- 
tion is  that  the  failure  of  the  serum  is  due  to  lack  of  complement : 
the  union  of  the  amboceptor  and  bacterium  is  supposed  to  take 
place  as  usual,  but  the  necessary  alexin  is  not  forthcoming.  This 
may  be  due  to  one  of  two  causes :  in  the  first  place,  there  may  be 
(as  is  known  to  occur  in  certain  diseases)  a  deficiency  in  the 
amount  of  complement  in  the  serum;  in  the  second  place,  that 
which  is  there  may  be  unsuitable  in  nature. 

As  regards  deficiency  in  complement,  this  has  been  found  to 
occur  in  certain  diseases,  and  is  very  probably  a  common  occur- 
rence in  pathological  conditions  and  states  of  malnutrition  in 
general ;  but  when  we  consider  the  comparatively  small  amount 
necessary  to  activate  a  large  dose  of  sensitized  bacilli,  there  is  no 
reason  to  think  that  it  ever  falls  below  that  level.  Again,  the 
facts  known  concerning  the  immunity  of  the  dog  and  other 
animals  to  anthrax  are  of  such  a  nature  as  to  render  it  improbable 
(in  this  case,  at  least)  that  deficiency  of  complement  can  really 
be  of  much  importance ;  for  dog's  serum  contains  abundance  of 
amboceptor,  yet  no  suitable  complement,  and  is  devoid  of  bacteri- 
cidal action.  We  shall  see  reasons  for  believing  that  amboceptor 
may  possibly  act  as  opsonin,  in  some  cases  at  least,  without  the 
concurrence  of  complement,  and  this  is  probably  the  explanation 
of  the  immunity  of  the  dog  to  anthrax. 

The  other  explanation,  that  of  Ehrlich,  is  that  the  complements 
present  in  human  serum  may  be  unsuitable  to  reactivate  serum 
derived  from  a  horse,  ass,  or  goat,  or  other  animal  used  as  the 
source  of  the  immune  body.  To  obviate  this,  he  has  proposed 
the  use  of  sera  from  several  species  of  animals,  in  the  hope  of 


BACTERIOLYSIS   AND    ALLIED    PHENOMENA  2OI 

finding  one  that  can  be  reactivated  by  human  complement,  and 
has  suggested  the  use  of  serum  from  the  higher  apes,  the  com- 
plements of  which  closely  resemble  those  of  man.  These 
explanations  are  not  very  satisfactory.  Thus  Shiga's  antidysentery 
serum  is  certainly  readily  complemented  by  human  blood;  and 
although  it  has  certainly  some  beneficial  action,  it  is  useless  in 
the  chronic  stages  of  the  disease,  and  this  although  the  amount 
injected  must  be  very  much  greater  than  is  necessary  to  dissolve 
all  the  bacilli  present  in  the  body. 

Another  possible  cause  of  failure  is  the  deviation  of  complement. 
If  we  admit  the  action  of  the  bactericidal  substances — by  no 
means  undisputed — in  the  natural  process  of  recovery  from 
disease,  we  can  easily  see  how  it  is  that  this  process  does  not 
occur  under  normal  conditions.  Thus,  when  infection  with  the 
typhoid  bacillus  occurs,  there  is  at  first  little  or  no  amboceptor 
in  the  blood.  The  small  amount  present  is  quickly  seized  by 
the  bacteria  and  removed,  and  although  a  few  bacilli  may  be 
killed,  the  great  majority  flourish  unchecked.  But  amboceptor 
is  soon  put  out  in  gradually  increasing  amounts,  and  at  first  is 
used  up  as  soon  as  it  is  formed.  The  two  processes,  proliferation 
of  bacilli  and  increase  in  the  amount  of  amboceptor,  now  progress 
side  by  side,  and  on  their  relative  rapidity  depends  the  outcome 
of  the  disease.  At  first  bacillary  proliferation  takes  place  more 
rapidly  than  the  production  of  antibody,  and  the  symptoms 
gradually  become  more  and  more  severe.  After  a  time  the 
antibodies  are  released  in  larger  and  larger  amount,  and  (in  a 
favourable  case)  a  time  arrives  when  there  is  exactly  enough 
for  all  the  bacteria  present.  We  must  assume  that  enough 
complement  is  available,  and  in  this  case  it  is  easy  to  see  how 
it  can  never  become  deviated ;  for  all  the  amboceptor  is  rapidly 
linked  up  to  the  bacilli,  and  does  not  accumulate  in  excess  in  the 
blood.  It  does  seem  possible,  however,  that  an  accumulation  of 
amboceptor  might  conceivably  determine  a  relapse,  bacteria 
which  escaped  destruction  owing  to  their  having  lain  in  the 
tissues,  gall-bladder,  or  other  inaccessible  region,  being  now  free 
to  grow  in  the  blood  owing  to  the  removal  of  complement  by 
deviation. 

But  when  a  dose  of  bactericidal  serum,  containing,  it  may  be, 
many  times  more  immune  body  than  is  necessary  for  the  solution 
of  the  bacilli  present,  is  suddenly  thrown  into  the  circulation, 
the  conditions  are  quite  different.  Here  there  is  an  excess  of 


2O2  FAILURE   OF    BACTERICIDAL    SERA 

immune  body  relatively  both  to  the  bacteria  and  to  the  com- 
plement, and  deviation  of  the  latter  may  occur.  Hence  it  is  at 
least  conceivable  that  a  dose  of  bactericidal  serum  may  be 
injurious  in  that  it  actually  inhibits  the  normal  bacteriolytic 
processes  that  are  at  work  in  the  blood-stream.  We  have  already 
quoted  processes  exactly  parallel  in  describing  the  experimental 
proof  of  the  deviation  of  complement. 

Another  suggestion  that  has  been  made  is  to  use  perfectly  fresh 
immune  serum,  or  to  reactivate  it  by  fresh  serum  from  a  normal 
animal.  But  this  seems  not  to  be  successful,  and  apparently 
alien  complements  rapidly  unite  with  the  tissues  of  the  animals 
into  which  they  are  injected,  and  so  become  inert. 

It  would  seem  that  no  explanation  based  on  deficiency  in  com- 
plement will  be  found  satisfactory :  the  facts  concerning  the 
action  of  the  dog's  serum  on  anthrax  bacilli  appear  to  offer  a 
crucial  experiment  settling  this  point.  Nor  if  the  added  ambo- 
ceptor  really  acts  as  opsonin  would  the  question  of  complement 
come  in.  The  most  satisfactory  explanation  appears  to  be  that 
the  sera  do  not  actually  come  in  contact  with  the  bacteria  in  the 
lesions,  though  they  may,  and  very  probably  do,  tend  to  sterilize 
the  blood,  and  so  prevent  further  generalization  of  the  infection. 
This  question — of  the  accessibility  of  the  bacteria  in  the  lesions 
to  the  substances  circulating  in  the  blood — is  probably  one  of  prime 
importance  in  immunity  and  recovery,  and  we  shall  meet  with  it 
again  in  dealing  with  the  opsonins.  It  seems  to  meet  the  facts 
of  the  case  very  well  with  regard  to  the  action  of  the  serum  in 
dysentery.  In  acute  cases  it  is  of  value ;  and  here  the  bacilli  are 
lying  in  regions  which  are  fairly  accessible  to  the  blood.  In  chronic 
dysentery  it  is  almost  useless,  and  in  this  form  of  the  disease  the 
bacilli  are  shielded  by  a  dense  and  impermeable  layer  of  inflam- 
matory tissues.  And  in  cholera  the  bacilli  are  mostly  lying  in  the 
intestinal  tract ;  probably  a  few  do  gain  access  to  the  blood  and 
tissues,  and  are  immediately  destroyed.  In  typhoid  fever  the 
bacilli  are  found  in  the  blood  early  in  the  disease,  and  later, 
roughly  at  the  period  at  which  antibodies  make  their  appearance 
in  large  amounts,  they  disappear.  But  there  are  always  large 
numbers  in  the  lymph  glands  and  spleen,  regions  in  which  it  is 
almost  certain  they  are  shielded  from  the  action  of  the  blood. 
This  explanation  appears  far  more  satisfactory  than  any  depending 
on  deficiency  in  complement. 

If  the  bacteria  in  the  blood-stream  are  actually  dissolved  by  the 


BACTERIOLYSIS    AND   ALLIED    PHENOMENA  203 

added  bactericidal  substances,  a  new  danger  is  involved — that  of 
the  liberation  of  a  large  amount  of  endotoxin.  This  substance 
we  believe  not  to  be  liberated  when  bacteria  are  dissolved  within 
the  leucocytes,  but  to  be  set  free  when  extracellular  solution  takes 
place.  The  essential  fever  of  the  early  stages  of  typhoid  fever 
is  very  probably  due  to  the  endotoxin  set  free  by  the  solution 
of  the  bacilli  in  the  circulating  blood,  and  any  sudden  addition  to 
this  amount  occurring  before  the  tissues  have  become  immunized 
or  trained  to  produce  anti-endotoxin  may  be  fraught  with  danger. 
There  can  be  little  doubt  that  if  sero-therapy  has  any  future 
triumphs  in  store,  they  will  be  in  the  direction  of  the  production 
of  anti-endotoxins. 

The  various  antibacterial  sera  in  common  use  are  considered  in 
the  last  section. 


CHAPTER  VIII 
THE  AGGLUTININS 

AN  exceedingly  interesting  and  important  group  of  antibodies, 
which  were  discovered  by  Gruber  and  Durham  in  1896  (though 
their  effect  had  been  observed  by  Charrin  and  Roger  in  1889 
in  the  case  of  B.  pyocyaneus),1  are  called  the  agglutinins,  since 
they  have  the  power  of  agglutinating  their  antigens,  or  causing 
them  to  adhere  in  masses.  Their  effect  is  best  seen  after  the 
addition  of  the  serum  of  a  patient  convalescent  from  typhoid  fever 
(or  of  an  animal  which  has  been  injected  with  typhoid  bacilli) 
to  a  living  culture  of  the  organism.  The  bacilli,  which  at  first  are 
actively  motile  and  are  distributed  uniformly  throughout  the  fluid, 
first  lose  their  motility,  and  then  individuals  may  be  seen  to  move 
nearer  and  nearer  to  one  another,  until  they  come  into  close  contact. 
It  often  happens,  especially  in  weakly  agglutinating  sera,  that  this 
approach  of  two  bacilli  may  be  seen  to  occur  before  their  paralysis 
has  taken  place.  They  then  revolve  rapidly  round  a  common 
axis,  giving  the  observer  the  impression  that  they  are  united 
together  by  a  sort  of  invisible  link,  which  they  struggle  to  break. 
This  process  continues,  and  fresh  individuals  are  attracted  to  the 
groups,  until  at  last  all  the  bacilli,  instead  of  being  scattered  equally 
throughout  the  fluid,  are  collected  into  masses,  the  intervening 
fluid  being  free.  The  process  may  also  be  watched  with  the  naked 
eye,  and  the  emulsion,  which  is  at  first  uniformly  turbid,  will  be 
seen  to  lose  its  homogeneity,  and  take  on  a  finely  granular 
appearance.  This  at  first  can  only  be  realized  by  comparison 
with  a  control  specimen  to  which  no  serum  has  been  added,  but 
in  a  little  time  it  will  be  obvious  that  flocculi  of  bacilli  are  being 
formed,  and  that  between  these  flocculi  the  fluid  is  clearing.  Soon 

1  The  effect  had  also  been  observed  by  Metchnikoff  in  the  case  of  V.  Metchni- 
kovi  in  1891 ;  he  was  inclined  to  regard  it  as  a  general  phenomenon,  but  failed 
to  find  it  in  another  case.  Similar  appearances  had  also  been  seen  by  Issaeff 
in  1893. 

204 


THE    AGGLUTININS  205 

(if  the  emulsion  is  thick  enough)  all  the  organisms  will  be  found  to 
have  collected  into  a  single  mass  or  a  few  masses,  the  rest  of  the 
fluid  being  quite  clear.  Finally,  these  masses  will  sink  to  the 
bottom  of  the  vessel,  and  it  will  be  noted  that  if  the  bacilli  in 
the  control  specimen  also  sink  (as  happens  with  killed  organisms), 
the  masses  will  be  much  more  voluminous  than  the  deposit  of 
unagglutinated  bacteria.  A  microscopic  examination  of  the  de- 
posit in  the  two  cases  will  show  why  this  is.  In  the  deposit  of 
dead  bacilli  the  separate  rods  have  sunk  down  slowly,  and  have 
packed  themselves  closely  together,  and  will  be  found,  to  a  very 
large  extent,  to  lie  horizontally  side  by  side.  In  the  agglutinated 
mass  the  bacilli  point  indifferently  in  all  directions,  and  the  explana- 
tion suggests  itself  that  they  have  been  drawn  forcibly  together  by 
a  centripetal  force,  and  have  not  had  time  to  adapt  themselves  so 
as  to  take  up  as  little  room  as  possible.  A  result  of  this  is  that  it 
is  easy  to  distinguish  between  a  specimen  that  has  agglutinated 
and  one  in  which  the  bacilli  have  simply  settled,  even  although  the 
actual  occurrence  of  the  phenomenon  has  not  been  witnessed. 

The  reaction  is  given  with  the  serum  of  immunized  animals,  and 
is  a  general  one.  It  is  given  with  nearly  all  species  of  bacteria, 
though  to  a  very  different  extent  in  different  cases,  with  red  blood- 
corpuscles,  leucocytes,  and  with  cells  of  all  kinds.  The  occurrence 
of  motility  is  not  necessary  for  it,  and  dead  bacilli  will  clump 
almost  or  quite  as  well  as  living  ones.  The  reaction  is,  in  general, 
specific,  and  a  serum  which  is  strongly  agglutinating  as  regards 
one  species  of  organism  may  be  entirely  devoid  of  action  on  others. 
Hence  it  was  proposed  by  Gruber  and  Durham  as  a  test  for  the 
identification  of  bacteria,  and  is  of  great  value.  Thus,  when  a 
bacteriologist  has  isolated  a  culture  of  an  organism  resembling 
B.  typhosus  from  a  patient  suspected  of  having  typhoid  fever,  or 
from  a  sample  of  water  supposed  to  be  contaminated,  the  first  step 
in  the  identification  is  made  by  observing  whether  it  is  clumped  by 
a  serum  known  to  have  agglutinating  powers  over  typhoid  bacilli 
and  not  over  others.  Other  tests  are  necessary  for  its  complete 
identification,  but  these  are  slower,  and  for  some  purposes  un- 
necessary. The  clinical  diagnosis  of  cholera  by  means  of  cultures 
from  the  stools  is  carried  out  in  the  same  way,  and  sera  adapted 
for  either  purpose  can  be  obtained  commercially. 

The  reaction,  however,  is  not  an  absolutely  specific  one,  and  it 
is  found  that  a  given  immune  serum  may  clump  not  only  the 
culture  used  in  its  production,  but  also  those  of  closely  allied 


2C>6  SPECIFICITY   OF   AGGLUTININS 

species.  Thus  typhoid  serum  clumps  B.  coli,  the  paratyphoid  and 
paracolon  bacilli,  the  B.  psittacosis,  and  others.  This  is  called  a 
group  reaction,  and  is  of  profound  interest  in  classification.  It  is 
not,  as  might  be  thought,  a  hindrance  to  the  practical  application 
of  the  process  as  a  method  of  identification  of  the  nature  of  a 
culture,  since  it  is  found  that  the  action  is  exerted  much  more 
strongly  on  the  organism  used  for  the  immunization  than  on 
others.  This  is  determined  by  ascertaining  the  dilution  necessary 
to  bring  about  agglutination  in  a  certain  time  at  a  given  tempera- 
ture. For  example,  we  may  find  that  certain  specimens  of  anti- 
typhoid serum  will  agglutinate  typhoid  bacilli  at  a  dilution  of 
i  :  10,000  in  one  hour,  whilst  B.  coli  is  not  affected  if  the  dilution 
is  greater  than  i  :  50.  In  the  practical  use  of  this  serum  we 
should  not  be  certain  that  a  given  culture  was  one  of  B.  typhosus 
unless  it  reacted  at  i  :  1,000  or  more. 

The  explanation  of  these  group  reactions  on  Ehrlich's  theory 
offers  no  difficulties.  Agglutinin  is,  as  will  be  shown,  a  specific 
antibody  to  the  molecules  of  protoplasm  contained  in  the  bodies  of 
the  injected  cells.  In  each  cell  these  will  be  of  many  varieties, 
and  to  each  a  specific  antibody  will  be  produced.  We  must 
imagine  a  typhoid  bacillus  as  containing  a  large  number  of  one 
particular  sort  of  molecule,  a  smaller  one  of  another,  whilst  in  the 
colon  bacillus  these  relations  will  be  the  reverse.  A  typhoid  serum, 
therefore,  will  contain  much  agglutinin  which  acts  on  the  typhoid 
molecules,  and  a  little  which  acts  on  a  few  of  those  present  in 
B.  coli ;  it  will  agglutinate  the  former  strongly,  the  latter  feebly. 
But  the  colon  serum  will  contain  antibodies  to  a  few  only  of  the 
molecules  present  in  the  typhoid  bacilli,  and  will  clump  it  only  in 
strong  dilutions.1 

Agglutinins  are  formed,  as  we  have  seen,  as  the  result  of  the 

1  There  are  a  few  noteworthy  exceptions  which  have  been  recorded  to  these 
general  rules.  In  a  few  cases  of  tuberculosis  the  power  of  agglutinating 
B.  typhosus  has  been  seen  to  rise,  and  Park  has  quoted  a  case  in  which  an 
animal  immunized  against  staphylococci  increased  ^its  power  against  the  same 
bacillus  from  i  :  10  to  i :  160.  In  interpreting  these  results  we  must  always 
wonder  whether  they  might  not  be  explained  by  a  rise  in  the  sensitiveness  of 
the  culture  used.  But  this  objection  does  not  apply  to  the  observations  of 
Posselt  and  Sagasser,  who  obtained  an  agglutinin  which  acted  on  bacteria 
other  than  those  used  for  the  injection,  and  which  was  not  removed  from  the 
serum  by  these  bacteria.  And  some  cases  have  been  recorded  in  which  a 
serum  had  less  action  on  its  own  antigen  than  on  others.  All  these  exceptions 
are  rare  and  not  full)'  investigated,  and  do  not  affect  the  general  law. 


THE   AGGLUTININS  2O7 

injections  of  their  specific  antigens.  They  are  also  frequently 
present  apart  from  any  interference.  For  example,  normal  human 
serum  clumps  the  second  vaccine  of  anthrax  powerfully,  and  in 
most  cases  has  a  feeble  action  on  both  B.  typhosus  and  B.  coli. 
Horse  serum  is  very  rich  in  agglutinins,  clumping  typhoid  and 
coli  bacilli,  the  B.  pyocyarmts,  and  the  cholera  vibrio,  often  in 
dilutions  as  high  as  i  :  100.  In  most  cases  agglutinins  are  present 
in  small  amounts  in  the  serum  of  infants  and  young  children,  and 
become  more  abundant  in  later  life.  This  suggests  that  they  may 
be  formed — in  part,  at  least — by  a  process  of  auto-inoculation  with 
bacteria,  principally,  perhaps,  from  the  intestine.  We  have 
already  seen,  however,  that  on  Ehrlich's  theory  the  presence  of 
antibodies  in  normal  animals  is  readily  explicable  without  such 
assumption. 

The  injections  of  bacteria  or  cells  of  any  sort  leads  to  the  pro- 
duction both  of  agglutinins  and  of  cytolysins,  and  in  most  cases  of 
haemolysis  or  bacteriolysis  agglutination  occurs  as  the  first  step  in 
the  process.  The  question  arises,  therefore,  whether  they  are  the 
same  substance.  It  is  easy  to  show  that  they  are  not,  since  sera 
which  contains  agglutinin  do  not  necessarily  contain  immune  body, 
or  vice  versa.  In  sera  obtained  by  artificial  immunization,  of  course, 
the  two  are  almost  invariably  formed  side  by  side,  and  it  is  only 
by  special  processes  that  we  can  obtain  the  one  without  the  other. 
Thus  Frouin  claims  that  if  dried  dog's  corpuscles  are  washed  with 
acetone  and  injected  into  a  rabbit,  they  cause  the  production  of 
agglutinin ;  but  no  haemolysin.  The  residue  from  the  evapora- 
tion of  the  acetone,  on  the  other  hand,  yields  haemolysin,  but  no 
agglutinin.  But  in  sera  from  normal  animals  it  is  quite  common 
to  find  the  one  without  the  other.  Thus  the  serum  of  healthy 
human  beings  frequently  clumps  normal  human  corpuscles,  but 
haemolysis  is  extremely  rare.  The  converse  process — haemolysis 
without  agglutination — also  occurs;  and  with  regard  to  antibacterial 
sera  of  artificial  origin,  Frankel  and  Otto  found  that  when  a  dog 
was  fed  on  typhoid  cultures  it  developed  agglutinin,  but  no  immune 
body.  Lastly,  in  many  cases  the  action  of  agglutinin  is  destroyed 
at  a  lower  temperature  than  that  of  immune  body,  although  both 
substances  are  in  a  marked  degree  thermostable.  We  shall  have 
to  discuss  the  effects  of  heat  on  agglutinin  more  fully  subsequently. 

There  is,  as  a  matter  of  fact,  a  kind  of  antagonism  between 
agglutination  and  cytolysis.  Cells  which  are  crowded  firmly 
together  are  naturally  shielded,  more  or  less,  from  the  solvent 


208  AGGLUTININS   IN    IMMUNITY 

action  of  the  fluid  in  which  they  are  suspended  ;  and  equally 
naturally  cells  which  are  dissolved  do  not  show  ordinary  agglu- 
tination, though,  as  we  shall  see,  they  show  a  similar  phenomenon. 

The  formation  of  agglutinins  follows  laws  similar  to  those 
governing  the  formation  of  other  antibodies.  After  each  injec- 
tion there  is  a  negative  phase,  followed  by  a  rise,  which,  as  a  rule, 
attains  its  maximum  in  about  a  week.  In  the  case  of  typhoid 
fever  no  agglutinin  can  be  demonstrated,  as  a  rule,  during  the 
first  week ;  there  is  then  a  steady  rise,  which  usually  attains  its 
maximum  at  the  commencement  of  convalescence.  After  this 
the  amount  tends  gradually  downward,  and  disappears  after  a 
time,  which  varies  between  a  few  months  and  several  years. 
On  a  single  occasion  the  author  has  seen  a  marked  drop  in  the 
amount  precede  a  relapse,  during  which  a  second  rise  occurred. 
This  was  obviously  a  negative  phase,  and  the  occurrence  of  the 
relapse  might  have  been  foretold  therefrom. 

Bacteria  which  have  been  acted  on  by  agglutinin  are  not  altered 
thereby  in  appearance,  viability,  or  virulence,  and  the  process  does 
not  appear  to  play  a  part  of  much  importance  in  immunity.  Two 
suggestions  have  been  made  in  this  respect  :  Gruber  thought  it 
caused  the  outer  layer  of  the  bacillus  to  swell  up,  so  that  it  could 
be  attacked  by  alexin,  and  Walker  suggested  that  the  clumping 
of  the  bacilli  might  render  them  more  easily  taken  up  in  large 
numbers  by  the  leucocytes.  Possibly,  also,  the  paralysis  is  the 
essential  feature  of  the  process,  as  a  reaction  of  immunity,  since 
we  should  expect  non- motile  bacteria  to  be  more  easily  ingested 
by  phagocytes.  It  is  interesting  in  this  connection  to  notice  that 
the  bacteria  for  which  strong  agglutinating  sera  are  obtainable 
are  all  highly  motile  (B.  typhosus,  coli,  and  pyocyaneus,  vibrios). 
The  recent  researches  on  the  thermostable  opsonins  have  caused 
a  certain  amount  of  attention  to  be  directed  to  the  agglutinins 
from  this  point  of  view,  but  nothing  is  definitely  proved. 

That  agglutinin,  in  common  with  the  other  antibodies,  unites 
directly  with  its  antigen  may  be  shown  in  several  ways.  In  one 
an  agglutinating  serum  cooled  to  o°  is  added  to  a  culture  similarly 
cooled,  and  the  mixture  kept  on  ice.  The  bacteria  will  gradually 
settle  down  without  agglutinating,  and  the  supernatant  fluid  may 
be  pipetted  off.  This  may  be  tested  in  the  ordinary  way,  and 
will  be  found  to  have  lost  much  of  its  agglutinating  power.  The 
bacteria,  if  suspended  in  warm  saline  solution,  will  immediately 
clump.  Evidently,  therefore,  the  agglutinin  has  been  removed  in 


THE    AGGLUTININS  2Og 

combination  with  the  bacteria.  Further,  it  is  clear  that  we  may 
distinguish  two  properties  of  agglutinin  (that  of  uniting  with  antigen 
and  that  of  clumping),  and  that  these  are  discharged  at  different 
temperatures  :  the  agglutinin  unites  at  o°,  and  only  exerts  its 
specific  action  at  higher  temperatures.  We  may  express  this  in 
Ehrlich's  terminology  by  saying  that  it  possesses  a  haptophore 
group  which  functionates  at  o°,  and  an  ergophore  group  which 
only  acts  in  the  warm. 

Another  proof  is  as  follows  :  It  was  shown  by  Bordet  that 
agglutination  only  takes  place  when  certain  salts  are  present.  Of 
these  sodium  chloride  appears  to  be  the  most  generally  efficient, 
but  Crendiropoulo  and  Amos  have  shown  the  calcium  chloride 
has  a  special  adjuvant  action  in  the  agglutination  of  cholera 
vibrios.  To  this  subject  we  shall  return.  The  proof  of  the  union 
between  bacteria  and  their  agglutinins  is  made  as  follows : 
Bacteria  are  added  to  clumping  serum,  and  the  precipitate  collected 
and  washed  and  shaken  in  a  large  quantity  of  distilled  water. 
No  agglutination  occurs  until  salt  is  added,  when  it  takes  place 
rapidly,  according  to  the  thickness  of  the  emulsion.  In  this  case 
also  the  two  substances  must  have  entered  into  the  combination. 

The  substance  with  which  agglutinin  combines — i.e.,  that 
which  calls  forth  its  production  in  the  living  animal — is  evidently 
not  a  toxin,  since  an  agglutinating  serum  has,  as  such,  no  protec- 
tive action.  We  know  some  of  its  characters.  It  is  formed,  of 
course,  in  the  bodies  of  the  bacteria,  and  in  young  cultures  is 
entirely  intracellular.  In  older  cultures,  however,  it  diffuses  out, 
being  probably  set  free  by  a  process  of  autolysis,  and  passes  into 
solution.  This  is  especially  the  case  in  broth  cultures,  and  this  is 
one  of  the  reasons  why,  if  liquid  cultures  are  used  in  agglutination 
tests,  they  must  be  young ;  in  agar  cultures  there  is  less  diffusion 
of  the  agglutinable  substance,  and  the  need  is  not  so  great.  Its 
presence  may  be  proved  in  two  ways :  In  the  first  place,  this 
filtrate,  if  injected  into  animals,  will  bring  about  the  production 
of  agglutinin,  as  we  should  expect.  In  the  second  place,  this 
fluid,  when  added  to  a  powerful  clumping  serum,  will  cause  a 
precipitate.  This  is  Kraus's  reaction,  and  it  is  a  most  interesting 
phenomenon.  It  is  best  seen  when  the  fluid  portion  of  broth 
culture  of  B.  typhosns  or  V.  cholera  (at  least  a  month  old  and 
filtered  through  a  Berkefeld  filter  to  remove  all  solid  particles)  is 
added  in  various  proportions  to  a  strong  immune  serum.  Under 
such  circumstances  the  fluid  will  gradually  become  opalescent,  or 


210  AGGLUTINOID 

even  opaque,  then  granular,  and  finally  flocculent.  It  presents  a 
most  extraordinary  resemblance  to  the  clumping  of  an  ordinary 
culture,  but  a  microscopic  examination  will  show  the  flocculi 
consist  of  amorphous  granules  instead  of  bacteria.  It  has  been 
suggested  that  it  is  due  to  a  clumping  of  cilia  which  have  passed 
through  the  filter  (Nicolle),  but  the  phenomenon  has  since  been 
observed  in  the  case  of  the  pneumococcus  (Panichi)  and  other 
non-flagellated  organisms.  The  agglutinable  substance  is  thermo- 
stable. It  does  not  appear  to  be  given  off  in  all  cases,  and  some- 
times all  attempts  to  get  Kraus's  reaction  are  unsuccessful. 

This  substance  is  the  antigen  of  agglutinin,  and  our  nomen- 
clature would  be  more  uniform  if  we  were  to  call  it  agglutin  and 
its  antibody  anti-agglutin,  but  the  terms  are  too  firmly  fixed  to  be 
altered.  We  shall  call  it  agglutinable  substance,  or  agglutinogen. 

The  fact  that  heated  serum  still  agglutinates  shows  that  alexin 
or  complement  plays  no  part  in  the  process,  but  we  have  already 
explained  how  we  know  that  the  molecule  of  agglutinin  possesses 
an  ergophore  or  zymophore  group.  This  group,  as  is  the  case 
with  the  corresponding  groups  of  the  toxins  and  complements,  is 
less  resistant  than  is  the  haptophore  group,  and  is  destroyed  at 
70°  to  75°  C.  The  substance  left  is  called  agglutinoid,  and  is 
analogous  to  toxoid  and  complementoid.  Its  existence  is  demon- 
strated thus  :  Heated  serum  (or  serum  which  has  been  kept  for  a 
long  time)  is  added  to  a  culture  of  bacteria.  No  agglutination 
takes  place.  The  bacteria  are  then  centrifugalized  off  and  placed  in 
a  strongly  agglutinating  serum,  but  are  found  not  to  clump.  It 
is  evident,  therefore,  that  the  bacteria  have  their  receptors 
occupied  by  some  substance  which  prevents  the  union  of  the 
agglutinin.  The  agglutinoid  has  combined  with  the  agglutinogen, 
and  excludes  the  unaltered  agglutinin. 

In  some  cases  at  least  agglutinoids,  which  have  a  stronger 
affinity  for  bacteria  than  has  normal  agglutinin,  may  be  present. 
In  this  case,  if  bacteria  be  added  to  a  mixture  of  the  two  sub- 
stances, no  agglutination  occurs.  The  pro-agglutinoids  (as  they  are 
termed,  the  expression  being  taken  from  the  prototoxoids)  seize 
on  the  agglutinable  substance  in  the  bacteria  before  the 
agglutinin  can  do  so.  If  to  this  mixture  more  bacteria  be  added, 
more  pro-agglutinoid  will  be  taken  up,  until  it  is  all  exhausted, 
and  then  any  fresh  bacteria  that  are  added  will  be  clumped. 
This  is  one  explanation  of  a  phenomenon  which  is  fairly  frequently 
observed  (if  looked  for)  in  the  clinical  diagnosis  of  typhoid  fever, 


THE    AGGLUTININS  211 

and  is  probably  a  source  of  error  often  overlooked  :  the  serum 
clumps  at  a  high  dilution,  and  not  at  a  low  one.  The  author  has 
observed  it  three  times  in  the  last  four  years.  Another  explana- 
tion, which  is  probably  more  often  the  true  one,  is  that  in  the  low 
dilutions  partial  bacteriolysis  takes  place,  and  the  partly  dissolved 
bacteria  do  not  clump.  The  reason  for  this  conclusion  is  that  the 
clumping  may  occur  in  low  dilutions  in  the  cold,  when  bacterio- 
lysis does  not  take  place.  Yet  other  explanations  have  been 
given. 

Certain  non-specific  substances  may  bring  about  clumping 
which  has  a  close  superficial  resemblance  to  that  caused  by 
agglutinin.  This  was  first  showed  by  Malvoz  in  the  case  of  the 
action  of  chrysoidin  on  V.  cholera.  He  also  showed  that  certain 
stains,  such  as  fuchsin,  vesuvin,  and  safranin,  and  some  anti- 
septics, such  as  formalin  (in  fairly  large  amounts),  corrosive 
sublimate,  and  peroxide  of  hydrogen,  have  this  action.  Mineral 
acids  also  possess  this  property,  and  also  certain  salts.  In  the 
case  of  cholera  vibrios,  Ruffer  and  Crendiropoulo  found  calcium 
chloride  to  have  a  powerful  action,  sodium  phosphate  to  have  a 
very  slight  one.  This  must  not  be  confused  with  the  effect  of 
salts  in  favouring  the  action  of  agglutinating  serum. 

We  are  now  in  a  position  to  discuss  the  mechanism  of  the 
process.  Numerous  theories  have  been  propounded.  Thus 
Gruber  thought  that  the  external  membrane  of  the  bacterium 
became  "  sticky,"  so  that  organisms  once  brought  into  contact 
remained  adherent.  But  no  visible  alteration  of  the  organisms 
or  red  corpuscles  can  be  seen.  Further,  it  would  not  account  for 
the  approach  of  two  non-motile  cells,  which  certainly  appears  to 
take  place  in  clumping,  and  would  not  explain  why  the  cells  or 
bacteria  were  brought  into  contact  in  the  first  instance.  Nicolle 
propounded  a  similar  theory.  He,  however,  showed  that  when 
inert  and  insoluble  particles,  such  as  of  talc,  were  suspended  in  old 
filtered  cultures  of  typhoid  bacilli  (Kraus's  fluid),  and  serum 
added,  they  appeared  to  clump  just  as  typhoid  bacilli  did,  and  it 
is  difficult  to  reconcile  this  with  his  theory.  Dineur  thought  that 
the  flagella  of  the  bacilli  might  have  an  adhesive  material 
deposited  on  them ;  but  many  non -flagellated  bacteria  clump,  to 
say  nothing  of  red  corpuscles.  Others  have  thought  that  Kraus's 
reaction  is  the  fundamental  phenomenon,  and  that  the  bacteria, 
etc.,  are  entangled  in  it .  like  the  particles  of  talc  in  Nicolle's 
experiment.  But  no  obvious  precipitate  can  be  seen  in  stained 

14—2 


212  MECHANISM    OF    AGGLUTINATION 

films  of  clumped  bacteria,  whereas  Kraus's  precipitate  is  easily 
demonstrated ;  besides  which,  agglutination  can  be  perfectly 
easily  demonstrated  in  young  cultures  (the  fluid  portion  of  which 
will  not  precipitate  with  specific  serum)  or  with  carefully  washed 
bacteria.  This  explanation,  though  ingenious,  may  be  disre- 
garded, r 

Bordet's  view  is  undoubtedly  the  correct  one.(  It  explains  agglu- 
tination as  being  due  to  a  change  in  the  molecular  relations 
between  the  objects  and  the  fluids  which  bathe  it — in  other  words, 
it  is  practically  an  effect  of  surface  tension^  It  takes  place  in 
many  cases  other  than  those  in  which  it  is  produced  by  specific 
sera  acting  on  bacteria,  red  blood-corpuscles,  etc. :  thus,  these 
objects  can  be  made  to  clump  by  the  action  of  many  aniline  stains, 
acids,  antiseptics,  etc.  An  emulsion  of  clay  in  distilled  water  will 
remain  turbid  for  a  long  time,  but  will  rapidly  clear,  owing  to  the 
formation  of  aggregates  of  particles,  when  salt  is  added.  This 
phenomenon  (which  explains  the  formation  of  mudbanks  at  the 
mouths  of  rivers,  where  admixture  of  fresh  and  salt  water  occurs) 
is  of  especial  interest  in  view  of  the  necessity  for  the  presence  of 
salts  in  specific  agglutination.  Many  bacteria,  especially  the 
tubercle  bacillus,  clump  spontaneously  without  the  addition  of 
serum.  In  some  cases  this  can  be  avoided  by  using  a  fluid  poor  in 
or  free  from  salt  to  make  the  dilution,  as  in  Sir  Almroth  Wright's 
method  of  estimating  the  agglutinating  power  of  the  serum  on  the 
tubercle  bacillus.  A  process  fundamentally  similar  can  be  seen  if 
wooden  matches  smeared  with  grease  are  thrown  on  to  the  surface 
of  water,  and  may  also  be  seen  in  the  gathering  together  of 
bubbles  on  the  top  of  any  fluid. 

Two  phenomena  are  involved  :  the  approach  of  the  particles  the 
one  to  the  other,  and  their  adhesion  subsequently.  The  former 
depends  on  certain  physical  laws  investigated  by  Korn  and  others, 
and  not  yet  fully  elaborated,  in  virtue  of  which  two  elastic 
particles  suspended  in  an  inelastic  fluid  in  which  vibrations  are 
taking  place  tend  to  approach  one  another.  It  is  probably  fair  to 
assume  that  these  conditions  are  always  present  in  the  case  of 
bacteria  suspended  in  a  fluid  medium,  and  that,  even  in  the 
absence  of  any  agglutinin,  the  individual  organisms  will  tend  to 
approach  one  another  and  to  form  aggregates.  But  in  the  case 
of  most  organisms  the  aggregates  thus  formed  are  quite  instable, 
breaking  up  when  the  slightest  shaking  of  the  fluid  takes  place. 
Here  the  force  of  surface  tension  is  all-important.  It  is  a  force 


THE    AGGLUTTNINS  213 

which  is  generated  wherever  a  fluid  comes  into  contact  with  any 
other  substance,  whether  solid,  liquid,  or  gas,  and  which  acts 
exactly  as  if  the  surface  of  the  fluid  in  question  were  in  a  state  of 
tension,  like  a  stretched  film  of  indiarubber.  If  a  relatively 
small  amount  of  any  fluid  be  suspended  in  another  fluid  of  the 
same  specific  gravity  with  which  it  does  not  mix,  it  will  assume 
the  form  of  a  sphere  :  this  is  because  the  sphere  has  a  smaller 
surface  for  a  given  volume  than  any  other  solid  body,  and  the 
hypothetical  film  on  the  surface  continually  contracts  until  this 
figure  is  assumed.  Hence  leucocytes,  and  most  other  free  cells 
consisting  of  fluid  or  semi-fluid  protoplasm,  tend  to  assume  a 
spherical  form  when  in  a  resting  condition  ;  hence  also,  of  course, 
the  spherical  form  of  soap-bubbles,  oil-globules,  etc.  Now  consider 
the  case  of  two  spheres  acted  on  by  surface  tension  and  just 
touching  one  another ;  for  example,  take  two  drops  of  oil 
suspended  in  a  fluid  of  about  the  same  specific  gravity.  If  we 
regard  the  surface  of  the  two  spheres  as  continuous,  it  is  obvious 
that  it  is  much  larger  than  it  would  be  if  the  two  drops  coalesced 
to  form  a  single  sphere.  (It  is  roughly  larger  in  the  proportion  of 
4:3.)  The  film,  therefore,  will  contract  until  the  two  globules  are 
drawn  into  a  single  drop,  with  double  the  volume  of  each  original 
globule,  but  with  a  much  smaller  superficies  than  that  of  the  two 
separately.  This  process  will  take  place  whenever  two  bodies, 
neither  or  both  of  which  are  wetted  by  the  fluid,  are  brought  in 
contact  or  very  close  together :  when  one  is  wetted  and  the  other 
not,  they  tend  to  repel  one  another.  The  force  of  surface  tension 
only  extends  for  an  exceedingly  minute  distance  into  the  fluid 
from  the  surface,  and  therefore  does  not  draw  the  substances 
together  if  they  are  a  finite  distance  apart.  Its  action  comes  into 
play  when  the  two  bodies  touch  one  another  in  one  point,  so  that 
the  surfaces  between  the  two  bodies  and  the  fluid  join  to  become 
one  at  this  point.  Thus,  if  two  red  blood-corpuscles  touch  one 
another  obliquely  at  one  point,  they  become  drawn  together,  and 
slide  the  one  on  the  other  until  they  oppose  as  small  a  surface  as 
possible  to  the  surrounding  fluid.  This,  of  course,  is  when  the  one 
lies  flat  on  the  other,  as  in  rouleaux  formation.  Two  wooden 
discs  enclosed  in  a  small  indiarubber  bag  would  act  precisely 
similarly. 

The  exact  way  in  which  the  agglutinin  affects  the  surface 
tension  between  the  bacteria  and  the  fluid  in  which  they  lie  is  not 
quite  clear,  and  raises  difficult  questions  in  molecular  physics,  some 


214  MECHANISM    OF    AGGLUTINATION 

of  which  are  glanced  at  in  our  section  on  Colloidal  Chemistry.  It 
is  intimately  concerned  with  the  subject  of  solubility.  If  a  body 
is  soluble  in  a  fluid — i.e.,  if  the  molecules  of  the  latter  have  a 
greater  affinity  for  those  of  the  former  than  these  have  for  one 
another — there  will  be  no  sharp  line  of  demarcation  between  the  two  : 
between  the  solid  and  the  liquid  there  will  be  a  zone  in  which  mole- 
cules of  both  substances  are  present,  and  this  will  shade  gradually 
off  into  the  solid  body  on  the  one  hand  and  the  fluid  on  the  other. 
Here,  then,  there  will  be  practically  no  surface  between  the  two, 
and  surface  tension  will  be  small  or  absent ;  ..and  in  a  general  way 
substances  present  in  a  fluid  which  dissolves  them  have  no 
tendency  to  clump.  Thus  to  prepare  an  emulsion  of  an  oil,  a 
solution  of  a  soap  or  of  an  alkali  is  used,  and  the  emulsions  thus 
formed  are  comparatively  stable  ;  but  if  the  fluid  be  made  acid, 
the  surface  tension  is  increased,  and  the  globules  quickly  run 
together  or  clump.  Now  it  is  clear  from  the  fact  that  the  fluid 
part  of  bacterial  emulsions  will  give  Kraus's  reaction,  and  will 
lead  to  the  production  of  antibodies  on  injection,  that  a  certain 
amount  of  solution  does  take  place.  That  agglutinin  actually 
renders  the  bacteria  less  soluble  appears  clear  from  the  phenomena 
of  Kraus's  reaction,  though  here  the  insoluble  precipitate  is  formed 
on  and  in  the  bacteria,  rather  than  in  the  fluid.  And  the  complete 
absence  of  clumping  which  occurs  when  bacteriolysis  takes  place 
(though  there,  is  a  large  amount  of  agglutinin  in  the  serum  used) 
is  an  indication  of  what  takes  place  when  the  bacteria  are  rendered 
more  soluble,  instead  of  less,  by  means  of  an  antibody.  Insolubility 
does  not  account  for  the  whole  of  the  phenomena,  but  it  is  a  feature 
of  great  importance. 

As  regards  the  nature  of  agglutinin,  all  we  know  is  that  it  is 
precipitated  with  the  globulins,  and  may  be  of  that  nature.  It 
does  not  dialyze,  and  is  digested  by  trypsin,  etc. 

It  appears  to  be  formed  in  the  lymphoid  organs,  red  marrow, 
and  spleen,  being  found  early  in  those  organs  after  injections  of 
cholera  vibrios  (Pfeiffer  and  Marx).  Metchnikoff  found  that  the 
peritoneal  exudate  might  be  richer  in  agglutinins  than  the  blood, 
and  thought  they  came  from  the  cells  (leucocytes  and  endothelial) 
in  that  fluid.  The  subject  has  also  been  investigated  by  Van 
Emden,  Deutsch,  and  Ruffer  and  Crendiropoulo,  who  all  confirm 
Pfeiffer  and  Marx  as  to  the  early  presence  of  these  substances  in 
the  lymphoid  tissues  after  inoculation. 

So  far  the  study  of  the  agglutinins  has  not  presented  much 


THE    AGGLUTININS  215 

difficulty,  but  further  research  has  shown  it  to  be  full  of  com- 
plexities. We  will  glance  briefly  at  some  more  recent  researches 
on  the  subject,  the  exact  explanation  and  significance  of  which 
are  not  ascertained  beyond  dispute. 

Several  facts  go  to  show  that  the  agglutinin  of  B.  typhosus  is  not 
a  simple  substance,  but  that  two  or  more  bodies  are  concerned. 
(It  may  be  mentioned  that  this  bacillus  has  been  studied  more 
than  any  other  in  this  connection.)  Thus  Joos,  after  a  series  of 
ingenious  researches,  came  to  the  conclusion  that  the  bacillus 
contains  two  agglutinogens,  and  that  each  has  its  corresponding 
agglutinin.  The  agglutinogen  which  is  present  in  largest  amount 
(and  which  he  calls  a)  is  thermolabile,  being  destroyed  at  62°  C., 
leaving  only  agglutinogen  /3,  which  is  thermostable.  An  animal 
injected  with  living  cultures  will  contain  agglutinins  (a  and  /3) 
against  both  the  substances.  The  first  will  combine  with  agglu- 
tinogen a  only,  whilst  the  second  will  combine  with  both  substances. 
The  two  substances  differ  in  their  thermostability  :  a  is  thermo- 
stable, but  /3  loses  its  power  of  agglutination  at  62°  C.  A  couple 
of  examples  of  the  facts  which  this  complicated  theory  was  intro- 
duced to  explain  may  be  given.  The  serum  of  a  horse  treated 
with  living  typhoid  bacilli  (and  therefore  containing  agglu- 
tinins a  and  /3)  clumped  a  living  culture  at  i  :  20,000,  and  a 
heated  one  at  i  :  1,000.  When  the  supernatant  fluid  of  this  last 
dilution  was  tested  with  heated  typhoid  bacilli,  no  agglutination 
took  place  (agglutinin  /3  had  been  removed),  whereas  it  would 
clump  living  bacilli  readily  enough.  Agglutinin  a  was  present  in 
larger  amount  than  /3,  and  had  not  all  been  removed  at  this 
dilution.  Again,  when  heated  serum  is  added  to  heated  bacilli 
there  is  no  agglutination,  since  the  thermolabile  agglutinin  /3  is 
destroyed.  The  agglutinin  a,  it  is  true,  is  not  destroyed,  but  its 
agglutinogen  (which  is  thermolabile)  is.  But  when  living  bacilli 
are  now  added  clumping  occurs,  since  the  agglutinin  a  can  find 
unaltered  agglutinogen  a  to  affect. 

Smith  and  Reagh  (and  their  researches  have  been,  in  the  main, 
corroborated  by  others)  found  that  typhoid  bacilli  and  other 
flagellated  bacilli  might  form  two  agglutinins — the  one  acting 
on  the  agglutinogen  of  the  bodies  of  the  bacilli,  the  other  on  that 
of  the  flagella.  The  subject  has  also  been  investigated  in  a  some- 
what similar  way  by  Buxton  and  Torrey,  who  find  also  two 
agglutinins — the  one  to  a  substance  which  remains  attached  to  the 
body  of  the  bacillus,  whilst  the  other  can  be  separated  from  it  by 


2l6  THE    "  PRO-ZONES  "    IN    AGGLUTINATION 

a  temperature  of  72°,  followed  by  filtration.  The  action  of  the  two 
is  specific.  If  the  filtrate  be  injected  into  animals,  the  serum 
which  results  clumps  ordinary  typhoid  bacilli  well,  but  has  little 
action  on  those  from  which  the  separable  substance  has  been 
removed.  The  serum  obtained  by  injection  of  the  bacilli  deprived 
of  separable  substance  is  weaker,  and  has  an  equal  action  on  the 
bacilli  whether  normal  or  heated  and  deprived  of  soluble  substance. 

It  is  evident  that  the  subject  is  a  complicated  one,  and  this 
is  even  more  clear  from  the  researches  of  Dreyer  and  Jex- Blake 
on  the  agglutination  of  B.  coli  by  its  specific  serum.  Investigating 
first  the  behaviour  of  the  bacilli  when  heated,  they  found,  as  other 
observers  had  done,  no  alteration  at  60°  C.,  but  a  sudden  diminu- 
tion in  the  power  of  undergoing  agglutination  when  heated  to 
70°  C.  This,  of  course,  i<s  usually  ascribed  to  the  complete  or 
partial  destruction  of  the  agglutinogen,  though  this  explanation  is 
incomplete,  since  (as  Eisenberg  and  Volk  had  previously  found) 
the  bacilli  which  do  not  clump  will  still  combine  with  agglutinin. 

But  Dreyer  and  Jex-Blake  found  that  the  agglutinability  is 
partially  or  completely  restored  by  prolonged  heating  to  100°  C. 
After  exposure  to  this  temperature  for  a  period  of  from  two  to 
thirteen  hours,  the  susceptibility  of  the  bacilli  to  the  serum  might 
be  as  great,  or  almost  as  great,  as  at  first.  This  is  an  extra- 
ordinary fact,  and  one  for  which  no  adequate  explanation  is 
forthcoming.  The  only  parallel  is  the  behaviour  of  megatheriolysin 
(a  bacterial  haemolysin),  also  investigated  by  Dreyer.  This  is 
destroyed,  or  at  least  rendered  inert,  at  60°  C.,  and  reactivated  at 
the  boiling-point. 

These  authors  also  adduce  evidence  to  show  that  the  "  zones  of 
inhibition,"  or  "pro- zones,"  described  by  Eisenberg  and  Volk  as 
occurring  with  heated  serum,  cannot  be  explained  by  the  assump- 
tion of  the  presence  of  "  agglutinoids  "  with  a  high  affinity  for 
agglutinogen.  The  evidence  against  this  view  is  briefly  this :  If 
it  were  true,  the  more  the  serum  were  weakened  by  the  heat 
(i.e.,  the  greater  the  production  of  agglutinoid),  the  larger  should 
be  the  zone  of  inhibition,  and  vice  versa.  This  they  found  not 
to  be  the  case,  for  a  serum  which  had  not  been  appreciably  injured 
by  the  heat  might  have  a  large  zone  of  inhibition.  They  also 
found  exactly  similar  zones  in  investigating  agglutination  caused 
by  means  of  acids,  in  which  case,  of  course,  nothing  of  the  nature 
of  agglutinoids  could  occur.  Thus,  in  a  series  of  experiments  it 
was  found  that  when  orthophosphoric  acid  was  added  to  a  definite 


THE    AGGLUTININS  217 

volume  of  emulsion  of  B.  coli,  agglutination  occurred  when  between 
118  centigrammes  and  4  centigrammes  of  the  acid  was  present, 
and  when  between  i-i  milligrammes  and  0*001  milligramme,  but 
not  with  intermediate  amounts. 

Similar  phenomena  (i.e.,  the  presence  of  zones  of  inhibition)  can 
be  seen  in  many  of  the  actions  of  coagulants  on  colloid  emulsions 
or  solutions,  such,  for  instance,  as  the  precipitation  of  gum  mastic 
from  water  by  ferric  chloride  or  trisulphide  of  arsenic.  This 
case  also,  as  Dreyer  points  out,  shows  a  marked  analogy  with 
the  clumping  of  coli  bacilli  by  phosphoric  acid,  since  in  each  case 
the  zone  of  inhibition  becomes  smaller  when  the  agglutinating  sub- 
stance (bacilli  or  particles  of  gum)  become  more  numerous.  This 
analogy  between  the  agglutination  of  bacteria  and  the  flocculation 
of  colloids  has  been  investigated  by  Bechhold,  Neisser  and 
Friedemann,  and  Biltz,  and  is  a  subject  of  the  utmost  interest, 
and  one  which  bids  fair  to  revolutionize  our  views  on  the  inter- 
relations of  the  antigens  and  antibodies.  At  present  our  knowledge 
of  the  subject  is  hardly  sufficiently  advanced  to  justify  an  account 
of  the  experiments  and  theories  on  which  these  views  are  based, 
and  the  original  papers  must  be  consulted  for  further  information. 

To  revert  again  to  the  question  of  the  specificity  of  the  reaction  : 
The  subject  is  a  complex  one,  and  we  have  already  seen  that  the 
phenomenon  of  the  "group  reaction  "  leads  us  to  the  supposition 
that  bacteria  of  different  species  must  possess  molecules  of  the 
same  nature.  Further  study  shows  that  there  are  differences  in 
this  respect  between  bacteria  of  the  same  species,  which  are  in- 
distinguishable the  one  from  the  other  by  ordinary  morphological 
and  chemical  tests,  but  which  have  had  different  origins.  Thus  it 
is  found  that  if  the  serum  of  an  animal  which  is  strongly  immu- 
nized to  a  given  culture  of  V.  cholera:  be  tested  against  cultures  of 
various  stocks,  that  which  was  used  for  the  injections  will  be 
clumped  most  powerfully,  the  others  to  variable  degrees.  These 
apparent  anomalies,  though  inconvenient  in  practice,  tend  to  make 
us  regard  the  reaction  as  more  specific  rather  than  as  less — 
sharply  specific,  that  is,  as  regards  a  certain  sort  of  chemical 
substance  which  is  formed  in  greater  degree  by  certain  races  of  a 
given  species  than  by  others. 

This  modified  specificity,  sharp  as  regards  chemical  substances, 
but  not  as  regards  bacterial  species,  is  well  illustrated  in 
Castellani's  absorption  reaction.  If  an  agglutinating  serum  which , 
e.g.,  clumps  the  typhoid  bacillus  powerfully,  and  a  colon  less 


218 


CASTELLANl'S    ABSORPTION    REACTION 


strongly  (and  which  was  obtained  by  immunizing  an  animal 
against  typhoid)  be  saturated  with  typhoid  bacilli,  centrifugalized, 
and  retested,  it  will  be  found  that  when  the  serum  has  lost  its 
power  of  agglutinating  that  organism,  it  will  also  be  bereft  of 
power  over  the  B.  coli.  On  the  other  hand,  if  it  be  saturated 
with  colon  bacilli  until  all  its  agglutinating  power  on  that 
organism  is  removed,  its  action  on  B.  typkosus  will  be  intact  or 
but  slightly  reduced.  The  mechanism  by  which  this  is  brought 
about  is  readily  understandable  by  means  of  the  following  diagram 
(Bolduan). 

Fig.  i  represents  a  typhoid  bacillus,  shown  as  if  it  consisted  of 
three  varieties  of  proteid  material,  A,  B,  and  C,  of  which  A  is 
present  in  largest  amount.  The  agglutinin  formed  by  its  injection 
will  consist  of  three  substances,  each  specific  for  its  own  proteid, 
and  the  antibody  to  A  (which  we  may  call  the  main  agglutinin) 


B      C 


B 


Typhoid  Bacillus 


Colon  Bacillus 
E      H 


Dysentery  Bacillus 

FIG.  45.     (After  Bolduan.) 

will  be  most  abundant.  Fig.  2  represents  a  colon  bacillus,  and 
is  shown  as  consisting  of  four  forms  of  proteid,  of  which  B  is 
common  both  to  it  and  to  the  typhoid  bacillus.  It  follows  that 
the  antityphoid  serum  will  clump  this  bacillus  as  well  as  that  of 
typhoid,  though  not  in  so  high  a  dilution ;  the  serum  only  acts  in 
virtue  of  its  anti-B  agglutinin,  of  which  it  possesses  but  a  small 
amount,  and  this  can  only  act  on  proteid  B,  which  forms  but  a 
small  part  of  the  colon  bacillus. 

If  the  serum  in  question  be  saturated  with  typhoid  bacilli  the 
whole  of  the  agglutinins  are  removed  in  combination  with  the 
bacilli,  anti-B  amongst  them,  and  the  serum  will  become  devoid 
of  agglutinating  action  on  both  bacilli. 

But  if  it  be  saturated  with  colon  bacilli,  the  only  effect  will  be 
to  withdraw  the  anti-B  agglutinin  ;  the  agglutinins  to  A  and  C 
will  remain,  and  consequently  the  serum  will  only  lose  its  clump- 
ing power  on  the  typhoid  bacillus  to  a  slight  extent.  After 


THE    AGGLUTININS  2IQ 

saturating  the  serum  with  all  the  allied  bacteria  on  which  it  can 
act,  we  might — theoretically,  at  least — remove  all  the  partial 
agglutinins,  leaving  only  the  main  agglutinin,  which  acts  on  the 
proteid  formed  by  the  typhoid  bacillus,  and  by  it  only,  and  so 
obtain  a  truly  specific  clumping  serum. 

Castellani's  test  seems  to  be  generally  correct,  though  excep- 
tions have  been  recorded. 

Further,  bacilli  of  the  same  stock  can  be  made  to  vary  greatly 
in  their  sensitiveness  to  the  action  of  agglutinin  by  various 
methods.  These  have  been  carefully  studied  by  Bordet,  Nicolle, 
Kirschbruch,  and  others,  and  it  has  been  found  that  the 
sensitiveness  of  typhoid  bacilli  is  diminished  by  washing,  by 
culture  at  high  temperatures  (40°  C.)  or  low  temperatures,  by  the 
addition  to  the  medium  of  minute  traces  of  antiseptics,  and  is 
less  in  old  cultures  and  in  those  that  have  been  recently  isolated 
from  the  living  animal.  It  is  found  in  general  that  bacilli  just 
isolated  from  a  typhoid  patient  clump  badly,  and  that  they 
gradually  increase  in  sensitiveness  for  six  months  on  cultivation  in 
artificial  media :  a  very  faintly  acid  medium  is  the  most  suitable. 
Sometimes  a  culture  alters  very  rapidly  without  obvious  cause, 
and  this  is  a  possible  source  of  error  in  the  clinical  application  of 
Widal's  reaction.  The  author  in  one  case  found  a  culture  which 
was  clumped  by  a  certain  serum  at  i  :  60  was  clumped  by  the 
same  serum  at  i  :  250  three  days  later. 

Another  method  by  which  bacteria  can  be  made  to  diminish  in 
their  sensitiveness  to  clumping  is  by  cultivation  in  specific 
immune  serum.  This  was  first  observed  by  Ainley  Walker,  and 
since  it  is  of  some  theoretical  interest  in  connection  with  Welch's 
theory  of  the  nature  of  the  unknown  toxins,  requires  a  short 
description.  He  found  that  when  typhoid  bacilli  were  grown  in 
immune  serum  (of  course,  devoid  of  complement)  diluted  with 
broth,  they  gradually  lost  their  agglutinability,  and  became  more 
virulent.  Thus  both  of  the  effects  of"  passage  "  were  reproduced 
in  vitro,  and  Walker  further  found  that  bacilli  thus  treated  re- 
moved the  agglutinin  from  the  serum  in  which  they  were  grown, 
and  believed  his  results  could  be  best  explained  on  the  supposition 
that  the  bacilli  produced  specific  anti-agglutinins.  This,  of  course, 
tends  to  corroborate  Welch's  interesting  suggestion  that  some  of 
the  organisms  for  which  no  exotoxins  have  been  discovered  exert 
their  lethal  action  by  producing  antibodies  against  the  blood  of 
their  host. 


22O  IMMUNITY   OF    BACTERIA   TO    AGGLUTININS 

The  subject  has  been  investigated  by  others,  and  their  results 
do  not  altogether  corroborate  those  of  Walker.  Thus  Miiller 
found  the  same  inagglutinability  after  culture  in  diluted  typhoid 
serum,  but  did  not  find  any  evidence  .of  the  formation  of  an  anti- 
agglutinin  ;  on  the  contrary,  he  found  that  bacilli  thus  treated 
had  less  power  of  weakening  the  action  of  typhoid  serum  than 
had  normal  bacilli.  We  might  explain  his  results  by  saying 
the  bacilli  had  lost  their  agglutinable  receptors,  or  agglutinogen. 
Bail,  working  with  a  different  method,  obtained  comparable 
results,  though  he  explained  them  quite  differently.  He,  as  well 
as  Landsteiner  and  others,  noticed  that  bacilli  grown  in  the 
immune  serum  grew  into  long  branching  threads,  losing  their 
bacillary  character  entirely.  This  has  been  thought  to  be  a  sort 
of  end-to-end  agglutination. 

The  change  is  a  more  or  less  permanent  one.  Thus  Park  and 
Collins  found  that  cultures  (of  B.  coli  and  B.  dysenteric)  which 
had  lost  their  power  to  agglutinate  might  require  to  be  grown  for 
months  on  ordinary  agar  before  they  retained  their  normal  sensi- 
tiveness. The  change  is  evidently  a  very  profound  one,  and  one 
that  is  handed  on  for  many  generations. 

The  subject  has  recently  been  carefully  investigated  by  Marshall 
and  Knox,  working  with  B.  dysenteric.  They  found  the  same  loss 
of  agglutinability  when  the  bacillus  was  grown  on  immune  sera, 
but  the  same  change  also  occurred  when  normal  horse  serum  was 
used ;  and  they  further  showed  that  the  alteration  was  a  rapid 
rather  than  a  gradual  one,  as  Walker  had  found  in  the  case  of 
B.  typhosus.  With  regard  to  the  mechanism  of  the  process,  they 
proved  conclusively  that  the  modified  bacilli  had  no  power  of 
uniting  with  the  agglutinin,  as  Miiller  had  also  found.  It  is  quite 
clear,  therefore,  that  the  loss  of  agglutinability  is  due  to  the  loss 
of  appropriate  receptors,  in  this  case  at  least.  They  point  out, 
apropos  of  Welch's  hypothesis  of  toxins,  that  a  bacterium  which 
attempted  to  protect  itself  against  its  host  by  the  formation  of 
antibodies  would  have  but  a  poor  chance  of  surviving,  whereas 
by  the  simple  process  of  losing  some  of  its  receptors  tbey  can 
nullify  some  of  the  protective  mechanisms  possessed  by  the  latter. 

Their  explanation  of  the  process  by  which  these  resistant 
bacilli  are  produced  is  interesting  and  suggestive.  They  do  not 
think,  with  Walker,  that  the  bacilli  acquire  a  new  character — 
i.e.,  the  power  of  producing  antibodies — and  transmit  it  to  their 
descendants,  but  that  there  is  simply  a  process  of  natural  selection. 


Of 

«^£^ 

THE    AGGLUTININS  221 

In  any  culture  of  bacteria  it  will  be  found  that  some  individuals 
are  more  easily  agglutinated  than  others.  In  the  early  cultures 
in  clumping  serum  the  susceptible  forms  grow,  but  they  sink  to  the 
bottom  in  a  dense  network.  The  less  sensitive  forms  also  grow 
from  slight  granules  or  a  diffuse  turbidity  through  the  fluid.  If 
the  subcultures  are  inoculated  from  this  fluid,  the  change  to  the 
non -clumping  form  occurs  more  quickly  than  if  the  masses  were 
used  for  the  transfer.  There  is  therefore  a  process  of  selection 
of  the  non-clumping  forms,  and  in  time  all  the  susceptible  bacilli 
become  eliminated.  It  is  this  process,  though  in  a  more  marked 
form,  that  accounts  for  the  increased  virulence  of  the  bacteria, 
which  is  brought  about  by  passage.  For  here  all  bacteria  which 
are  not  resistant  to  the  bacteriolysins  and  to  the  phagocytic 
action  of  the  leucocytes  will  be  killed  off,  and  will  not  propagate 
their  species.  We  may  therefore  conclude  on  theoretical  grounds 
that  virulent  bacilli  are  those  having  (along  with  a  potent  toxin) 
few  receptors  which  can  be  attacked  by  amboceptor  and  opsonin. 
We  have  seen  that  Pfeiffer  and  Friedberger's  experiments  do 
not  tend  to  corroborate  this  (in  the  case  of  amboceptor),  but  that 
they  cannot  be  regarded  as  conclusive. 

The  increase  in  the  virulence  of  the  organism  by  cultivation  in 
immune  serum  has  been  generally  corroborated,  though  Roger 
found  the  opposite  to  occur  when  streptococci  were  cultivated  in 
antistreptococcic  serum. 

The  hamagglutinins  occur  in  normal  and  in  immunized 
animals.  Those  in  the  latter  call  for  no  especial  notice  ;  they  are 
developed  after  injection  of  red  corpuscles  side  by  side  with 
immune  body,  and  their  presence  can  be  demonstrated  if  the 
serum  be  heated  or  if  the  experiment  be  performed  in  the  cold. 
The  normal  agglutinins  which  one  species  may  possess  for  the 
red  corpuscles  of  another  species  call  for  no  special  notice. 

The  iso-agglutinins  are,  however,  worthy  of  a  short  description. 
They  are  common,  and  have  been  most  studied  in  human  blood, 
and  will  be  found  to  occur  to  a  greater  or  less  extent  in  most 
specimens  of  human  sera.  The  phenomena  they  produce  are 
entirely  similar  to  those  produced  by  a  specific  serum  in  a  typhoid 
culture.  Under  the  microscope  the  corpuscles  will  be  seen  to  run 
together  into  masses  which  are  quite  unlike  the  ordinary  rouleaux 
of  shed  blood  in  that  the  corpuscles  are  approximated  together 
without  any  trace  of  definite  arrangement.  When  a  strong  serum 
is  used  the  cohesive  force  may  be  so  great  that  the  corpuscles 


222  THE    H^MAGGLUTININS 

become  absolutely  fused  together  into  a  solid  mass,  in  the  centre 
of  which  the  outlines  of  the  original  corpuscles  cannot  be  made 
out.  The  macroscopic  appearances  are  similar  to  those  of  a 
Widal's  reaction,  the  emulsion  of  corpuscles  becoming  granular 
and  flocculent,  and  clear  rapidly,  with  the  deposition  of  a  red  mass 
at  the  bottom  of  the  vessel.  With  a  powerful  serum  a  column  of 
emulsion  2  inches  high  may  be  completely  cleared  in  five  minutes, 
and  the  clumping,  as  observed  in  a  drop  of  the  fluid  on  a  slide, 
may  be  complete  in  a  few  seconds. 

It  will  be  found  that  a  certain  serum  does  not  clump  all  human 
corpuscles  equally  well,  and  facts  have  been  observed  quite 
comparable  to  those  found  by  Ehrlich  in  the  case  of  isohsemo- 
lysin.  According  to  Landsteiner  (whose  researches  have  been 
corroborated  by  Hektoen),  human  bloods  may  be  divided  into 
three  groups,  which  Hektoen  describes  as  follows  : 

Group  i. — Here  the  corpuscles  are  not  agglutinated  by  sera  of 
the  other  groups,  whilst  the  sera  agglutinate  the  corpuscles  of 
both  groups. 

Group  2. — In  this  group  the  corpuscles  are  agglutinated  by  the 
sera  of  the  other  groups,  whilst  the  sera  agglutinate  the 
corpuscles  of  Group  3,  but  not  of  Group  i. 

Group  3. — The  corpuscles  are  agglutinated  by  all  other  sera, 
and  the  sera  agglutinate  the  corpuscles  of  Group  2,  but  not  of 
Group  i. 

A  few  specimens  of  blood  are  found  which  do  not  fit  exactly 
into  any  of  these  categories. 

It  will  be  noted  that  in  none  of  these  groups  is  there  an 
agglutination  of  red  corpuscles  by  its  own  serum — i.e.,  an  example 
of  the  presence  of  an  auto-agglutinin.  This  substance,  however, 
may  occur  in  the  blood,  but  apparently  only  in  disease.  The 
best  example  (in  fact,  the  only  one  in  which  it  has  been  positively 
demonstrated)  is  in  pernicious  anaemia.  In  this  disease  it  often 
happens  that  the  washed  corpuscles  are  immediately  and  strongly 
clumped  by  the  serum  of  the  same  patient,  and  the  same 
phenomenon  may  occur  when  citrated  plasma  is  used  instead  of 
serum.  Whether  similar  phenomena  occur  in  the  body,  and  if 
not,  the  reason  for  its  absence,  is  quite  unknown  :  it  is  exceedingly 
difficult  to  imagine  that  clumps  similar  to  what  we  see  in  vitro 
can  be  formed  in  the  circulation,  for  they  appear  to  be  much  too 
large  to  pass  through  the  capillaries.  The  only  plausible  explana- 
tion is  that  the  auto-agglutinin  does  not  exist  as  such  in  the 


THE    AGGLUTININS  223 

circulation,  only  coming  into  existence  as  the  blood  is  shed.  If 
this  is  the  case,  it  appears  clear  that  its  production  is  not  the 
result  of  clotting,  since  -the  clumping  may  occur  before  coagula- 
tion has  taken  place ;  indeed,  if  the  blood  is  carefully  watched  as 
it  flows  from  a  skin  puncture  in  a  marked  case  of  pernicious 
anaemia,  it  maybe  seen  to  become  "  streaky,"  pale  and  dark  areas 
being  present,  and  a  microscopical  examination  will  show  that 
this  is  due  to  clumping  of  the  corpuscles,  which  thus  appears  to 
take  place  immediately  the  blood  leaves  the  vessels. 

The  researches  of  Gay  would  appear  to  show  that  the  clumping 
of  the  red  corpuscles  by  serum  is  not  necessarily  due  to  the 
presence  of  an  agglutinin  at  all,  but  may  be  caused  by  variations 
in  the  tonicity  of  the  corpuscles  and  serum.  Thus  he  found  that 
in  the  bloods  which  have  non-agglutinable  corpuscles  there  is  a 
higher  molecular  concentration  than  in  the  other  groups,  indicating 
a  greater  amount  of  salts  both  in  the  corpuscles  and  in  the  sera. 
There  are  also  differences  in  tonicity,  though  less  marked,  between 
the  members  of  the  other  groups.  He  also  finds  that  if  a  serum 
which  is  hypertonic  to  a  certain  sample  of  corpuscles,  and  which 
therefore  clumps  them,  is  examined  after  contact  with  the 
corpuscles  in  question,  its  tonicity  is  decreased  until  it  reaches 
that  of  the  serum  which  normally  accompanies  those  corpuscles, 
which,  of  course,  it  now  no  longer  agglutinates.  This  is  a  simple 
saturation  experiment,  the  old  explanation  of  which  would  have 
been  that  the  agglutinin  had  all  been  removed  by  contact  with  the 
corpuscles,  but  which  Gay  explains  by  absorption  of  salts  by  the 
corpuscles.  Lastly,  a  simple  hypertonic  solution  of  NaCl  and 
CaCl2,  and  according  to  Peskind  a  large  number  of  acids  and  acid 
salts,  gave  rise  to  appearances  suggestive  of  clumping,  though  not 
identical  therewith.  His  researches  are  highly  suggestive,  and  it 
may  be  that  we  shall  have  to  modify  our  ideas  of  the  normal 
iso-agglutinins  ;  but  the  subject  requires  further  investigation. 

Rouleaux  formation  ]  presents  some  resemblances  and  also  some 
differences.  Red  blood-corpuscles,  when  washed  free  from  serum 
and  suspended  in  normal  saline  solution,  lie  free  side  by  side,  and 
show  no  tendency  to  run  together  ;  but  if  placed  in  human  serum 
and  some  other  fluids,  the  biconcave  discs  approach  one  another  in 
a  peculiar  orderly  fashion,  so  as  to  form  adherent  rolls  resembling 

1  I  am  indebted  to  some  unpublished  researches  of  Dr.  Wiltshire's  for 
much  interesting  information  on  this  subject.  Any  new  fact  mentioned  is 
owing  to  him,  unless  the  contrary  is  stated. 


224  ROULEAUX    FORMATION 

piles  of  money.  This  phenomenon  has  attracted  a  good  deal  of 
attention,  though  less  than  it  deserves,  and  the  exact  mechanism 
by  which  it  is  produced  is  still  uncertain.  It  has  obviously  some 
resemblance  to  agglutination.  It  is  quite  conceivable,  for  example, 
that  an  agglutinating  serum,  when  greatly  diluted,  might  act  so 
weakly  that  the  corpuscles  might  be  drawn  gradually  together, 
and  so  approach  one  another  in  such  a  fashion  as  to  form  the 
peculiar  rolls,  very  much  as  in  the  orderly  deposition  of  molecules 
to  form  a  crystal,  which  occurs  when  a  strong  solution  of  a  salt  is 
slowly  evaporated,  so  as  to  allow  the  molecular  attractions  to 
come  into  play  in  a  regular  fashion.  But  this  is  not  the  case, 
since,  as  was  shown  by  Descatello  and  Sturli,  it  is  not  possible  to 
dilute  an  agglutinating  serum  with  normal  saline  solution  so  as  to 
convert  it  into  a  rouleaux-inducing  one.  And  according  to  Lange, 
an  agglutinating  serum  which  has  had  its  agglutinin  removed  by 
saturation  with  corpuscles  will  still  give  rise  to  the  phenomenon 
under  discussion. 

Both  the  corpuscles  and  the  fluid  in  which  they  are  suspended 
require  consideration  in  the  discussion  of  the  question  why 
rouleaux  formation  occurs  in  shed  blood,  though  not  in  the  tissues. 
Human  serum  will  always  cause  it,  though  its  potency  in  this 
respect  varies  greatly  from  time  to  time  in  the  same  person,  and 
also  in  different  individuals  (as  tested  on  the  same  specimen  of  red 
corpuscles).  This  power  is  quickly  destroyed  on  dilution,  being 
almost  always  annulled  on  the  addition  of  an  equal  quantity  of 
normal  saline  solution.  The  roulogenous  principle  also  occurs  in 
human  milk,  but  not — or  not  commonly — in  that  of  cows,  and  is  not 
destroyed  by  boiling  for  five  minutes.  Viscosity  appears  to  play 
some  part  of  secondary  importance,  and  not  clearly  defined : 
solutions  of  colloids,  such  as  gum  or  gelatin,  may  induce  it,  as 
was  pointed  out  by  Wharton  Jones.  The  roulogenous  substance 
occurs  to  a  very  large  extent  in  inflammatory  exudates,  but  not  in 
transudates. 

It  is  possible  that  the  induction  of  rouleaux  formation  may  be 
due  to  the  development  of  some  substance  in  the  serum  at  the 
moment  the  blood  is  shed,  and  it  is  also  possible  that  it  may  be 
due  to  some  change  in  the  red  corpuscles.  It  has  been  shown 
that  biconcave  discs  suspended  in  water  and  smeared  with  grease 
will  run  together  in  this  way,  and  Brunton  has  suggested  that 
when  the  blood  is  shed  some  fatty  acid  is  liberated  by  the  action 
of  carbon  dioxide.  It  is  also  possible  that  the  change  which 


THE    AGGLUTININS  225 

occurs  when  the  blood  is  shed  may  be  one  of  shape.  Thus 
Weidenreich  and  others  hold  that  red  corpuscles  are  normally 
bell-shaped,  and  that  the  moment  they  leave  the  vessels  they 
become  biconcave  discs.  If  there  is  an  increase  in  the  tension 
generated  at  their  surface  of  contact  with  the  serum,  the  con- 
ditions for  rouleaux  formation  would  appear  to  be  present. 
Weidenreich's  facts  have  been  strongly  disputed,  and  are  not 
generally  accepted. 

Rouleaux  formation  cannot  be  induced   in   corpuscles  which 
have  been  heated  to  44°  C. 


CHAPTER  IX 
THE   PRECIPITINS 

AGGLUTININS  are  antibodies  obtained  by  the  injection  of  particu- 
late  substances,  and  have  the  property  of  causing  these  substances 
to  collect  into  clumps.  In  exactly  the  same  way  proteids  in  solu- 
tion, when  injected  into  suitable  animals,  bring  about  the  formation 
of  another  class  of  antibodies,  which  possess  the  power  of  clumping 
the  molecules  of  the  proteid  in  a  solution  similar  to  that  injected. 
This  manifests  itself  by  the  formation  of  a  precipitate.  After  the 
addition  of  the  clear  antiserum  to  the  clear  proteid  solution,  the 
mixture  becomes  opalescent,  and  then  opaque,  and  after  a  time  a 
precipitate  is  cast  down,  leaving  a  clear  supernatant  fluid.  Hence 
these  substances  are  called  precipitins,  the  substance  with  which 
they  form  a  precipitate,  and  which  calls  them  into  existence  when 
injected  into  a  suitable  animal  (their  antigen),  being  termed  pre- 
cipitable  substance,  or  precipitogen,  and  the  insoluble  combination 
of  the  two,  precipitum.  They  are  in  all  respects  closely  allied  to 
\  agglutinins,  if  not  absolutely  identical.  The  fact  of  their  acting  in 
V  a  clear  fluid  does  not  prove  that  they  exert  their  effect  in  a  true 
solution,  since  modern  physico-chemical  research  has  shown  that 
proteids  do  not  form  solutions,  but  merely  emulsions  or  suspensions 
of  molecules  or  of  complexes  of  molecules.  The  effect  of  the 
addition  of  a  precipitin  is  to  cause  an  agglutination  of  these 
molecules,  which  is  entirely  analogous  with  the  agglutination  of 
typhoid  bacilli.  The  laws  which  govern  the  reactions  of  the 
precipitins  and  agglutinins  are  entirely  similar,  and,  theoretically, 
it  would  probably  be  more  accurate  to  consider  them  under  one 
head.  The  practical  applications  of  the  two  classes  of  antibodies 
are,  however,  very  different,  and  it  is  more  convenient  to  treat 
them  as  separate  substances. 

The  first  substances  of  this  group  to  be  discovered  were  the 
bacterio  -  precipitins  of  Kraus,  first  investigated  in  1897,  anc^ 
referred  to  elsewhere.  Kraus  found  that,  if  he  took  an  old 

226 


THE    PRECIPITINS  227 

culture  of  typhoid  bacilli  and  filtered  it  to  remove  the  bodies  of 
the  bacteria,  and  then  added  some  typhoid  serum  to  the  clear 
solution,  a  precipitate  was  formed.  The  same  happened  with 
cultures  of  cholera  and  plague  organisms,  after  addition  of  the 
appropriate  sera,  so  that  the  reaction  is,  within  limits,  specific. 
The  limits  of  the  specificity  are  not  yet  thoroughly  ascertained, 
but  there  are  distinct  evidences  of  group  reactions  similar  to  those 
seen  in  the  agglutinins.  Thus  Norris  found  a  common  precipitin 
for  organisms  of  the  cholera  group,  for  some  cocci,  etc.  There 
were,  however,  some  exceptions  ;  thus,  a  rabbit  which  had  been 
injected  with  cultures  of  B. prodigiosus  developed  a  precipitin  which 
acted  on  filtered  cultures  of  B.  coli  and  V.  Metchnikovi,  as  well  as 
on  the  filtrate  from  the  organism  injected.  He  thought,  however, 
that  the  reaction  is  a  more  intimate  and  constant  test  of  group 
relationships  than  is  agglutination  ;  thus  he  prepared  an  antiserum 
to  a  bacillus  belonging  to  the  hog-cholera  group  which  did  not 
agglutinate  typhoid  or  colon  bacilli,  but  which  gave  the  precipitin 
reaction  with  their  filtrates.  The  bacillus,  it  may  be  pointed  out, 
is  a  member  of  the  same  group  as  the  other  two,  so  that  (in  the 
case  of  this  particular  serum)  the  agglutination  reaction  is  mis- 
leading. 

Bacterio-precipitins  may  be  prepared  by  the  injection  of  cultures 
of  the  bacteria,  or  the  filtrates  from  the  old  cultures  spoken  of 
above  ;  it  is  evident,  therefore,  that  they  are  antibodies  to  substances 
in  solution.  Some  authorities,  it  is  true,  have  thought  that  the 
actual  antigen  and  precipitable  substance  consist  in  reality  of  the 
broken-off  flagellae,  which  are  sufficiently  fine  to  pass  through  a 
Berkefeld  filter ;  but  this  can  hardly  be  the  case,  since  a  bacterio- 
precipitin  can  be  obtained  for  non -flagellate  organisms.  It  is  true 
that  the  best-known  and  most  powerful  of  these  antibodies  are  for 
organisms  which  possess  flagellae,  and  it  is  quite  probable  that  the 
clumping  of  these  filaments  does  occur  in  some  cases,  and  intensi- 
fies, or  may  be  mistaken  for,  a  true  precipitation. 

Bacterio-precipitins  cannot  be  (or  have  not  been)  prepared  to  all 
organisms.  Thus,  diphtheria  antitoxin  does  not  form  a  precipitate 
with  diphtheria  toxin,  a  substance  prepared  on  lines  exactly  similar 
to  those  used  in  procuring  the  precipitating  solution  for  typhoid 
and  cholera.  Diphtheria  toxin  is  an  excreted  substance,  not  a 
substance  dissolved  out  of  the  bacterial  protoplasm. 

The  first  to  observe  precipitins  to  other  proteid  solutions,  and  in 
particular  to  serum,  was  apparently  Tchistovitch,  in  1898.  He 

15—2 


228  PRECIPITOID 

investigated  the  formation  of  an  antitoxin  for  eel  serum  by  inject- 
ing that  substance  into  rabbits,  and  found  the  serum  thus  obtained 
not  only  neutralized  the  toxic  effects  of  the  eel  serum,  but  formed 
a  precipitate  with  it.  In  an  exactly  similar  way,  he  prepared  a 
precipitating  serum  to  a  non-toxic  serum — that  of  the  horse — and 
investigated  its  properties.  He  found  that  the  precipitate  formed 
by  the  interaction  of  the  antiserum  and  its  antibody  was  soluble  in 
dilute  acids  and  alkalis,  but  insoluble  in  water,  alkaline  carbon- 
ates, and  neutral  salts.  These  results  were  corroborated  by  Bordet, 
whose  classical  researches  on  the  haemolysins  were  made  about 
this  time.  He  found  that  the  injection  of  denbrinated  fowl's  blood 
into  rabbits  caused  the  appearance  of  agglutinins,  haemolysins,  and 
of  precipitins  in  the  serum  of  the  latter,  so  that  the  fowl's  red 
corpuscles  would  be  first  clumped  and  then  laked,  after  the 
addition  of  the  immune  serum,  and  this  substance,  when  added  to 
fowl  serum,  led  to  the  formation  of  a  precipitate.  Bordet  also 
demonstrated  the  formation  of  a  precipitating  serum  for  milk 
(lacto- serum).  These  researches  were  corroborated  by  Myers, 
Uhlenhuth,  Wassermann  and  Schiitze,  Nuttall,  and  others,  and 
the  reaction  has  now  been  very  fully  studied,  and  found  to  be  of 
considerable  practical  importance. 

Precipitins  are  in  all  respects  closely  analogous  to  the  agglutinins. 
They  are  formed  under  the  same  conditions — i.e.t  as  a  reaction  of 
the  cells  to  a  foreign  material,  probably  in  all  cases  of  a  proteid 
nature — and  appear  themselves  to  be  proteids.  They  are  destroyed 
by  pepsin  and  other  proteolytic  enzymes,  and  by  acids  and  alkalis. 
They  appear  to  be  allied  to  or  carried  by  the  globulins.  When 
heated  they  appear  to  undergo  a  change  into  precipitoid,  a  sub- 
stance which  has  the  power  of  combining  with  the  molecules  of 
precipitogen,  but  which  does  not  precipitate  them  ;  this  is  shown 
by  the  fact  that  a  further  addition  of  precipitin  does  not  cause 
precipitation,  indicating  that  the  effect  is  not  due  to  a  mere 
weakening  of  the  substance  or  to  its  partial  destruction.  Hence 
we  deduce  that  the  precipitin  molecule  consists  of  two  portions — 
a  thermostable  haptophore  or  combining  group,  and  a  thermolabile 
functionating  group,  the  action  of  which  is  necessary  for  agglutina- 
tion of  the  molecules  to  occur.  This  change  takes  place  at  a 
temperature  of  50°  to  60°  C.,  but  it  also  occurs  slowly  when 
precipitin  solutions  are  kept  at  ordinary  temperatures,  so  that  these 
become  gradually  useless  as  tests  for  their  antigens ;  and  the 
change  may  be  hastened  by  the  action  of  light  or  of  various 


THE    PRECIPITINS 


22g 


chemical   substances.     Precipitating   sera   should,   therefore,    be 
kept  in  a  dry  state  in  a  cool  place,  and  preserved  from  light. 

Precipitoids  appear  to  have  a  stronger  affinity  for  the  precipitate 
substance  than  has  the  unaltered  precipitin — an  alteration  in 
affinity  similar  to  that  which  we  have  seen  to  occur  sometimes  in 
the  case  of  agglutinin,  and  which  leads  to  the  formation  of  what 
we  have  termed  pro-agglutinoid.  Thus,  if  a  serum  which  has 
been  heated  until  it  has  lost  its  precipitating  power  be  mixed 
with  unheated  precipitin,  the  mixture  will  not  form  any  precipitate 
after  the  addition  of  small  amounts  of  the  normal  serum  ;  it  is  only 
after  enough  of  the  latter  has  been  added  to  combine  with  all  the 
precipitoid  that  a  precipitate  begins  to  appear.  The  same  pheno- 
mena may  occur  in  working  with  old  and  degenerated  sera,  or 
occasionally  even  with  fresh  ones.  An  example  will  make  this 
clear. 


Normal  Serum. 

Antiserum. 

Amount  of  Precipitate. 

7 

nil 

6 

nil 

5 

nil 

4 

nil 

3 

nil 

2 

0*5 

I 

I 

i 

I 

2 

1-25 

I 

3 

2 

I 

4 

3 

I 

5 

3  '5 

I 

6 

375 

7 

4 

8 

4 

10 

3-25 

12 

275 

H 

2-25 

16 

2 

18 

1-5 

20 

I 

24 

nil 

3° 

nil 

Here  the  maximum  precipitate  was  given  when  8  parts  of  anti- 
serum  were  mixed  with  i  part  of  normal  serum.  When  i  part  of 
normal  serum  was  mixed  with  24  parts  of  antiserum,  there  was  no 


230  PRECIPITOID 

precipitate — i.e.,  the  precipitoids  were  at  this  point  just  saturated  ; 
but  in  a  mixture  of  20  parts  of  antiserum  with  i  part  of  normal 
serum,  it  appears  that,  after  all  the  molecules  of  precipitoid  were 
saturated,  there  was  still  enough  precipitable  substance  present  to 
combine  with  some  of  the  precipitin,  and  thus  to  form  a  precipitate. 
In  the  mixture  of  8  and  i  the  precipitoid  was  all  saturated,  and  a 
maximum  amount  of  precipitable  substance  left  over  to  combine 
with  precipitin. 

This  theory  of  the  "  specific  inhibition  "  of  the  action  of  this 
precipitin  was  the  first  explanation  to  be  brought  forward,  and 
appears  to  afford  a  fairly  satisfactory  explanation  of  the  facts 
observed.  It  is,  however,  quite  probable  that  future  physico- 
chemical  research  may  show  it  to  be  erroneous,  and  that  these 
zones  of  inhibition  are  in  reality  similar  to  those  observed  in  the 
non-specific  precipitation  of  colloids,  of  which  we  have  already 
spoken.  To  this  subject  we  shall  return.  Be  the  explanation 
what  it  may,  the  phenomena  are  of  considerable  practical  impor- 
tance in  the  application  of  the  precipitating  sera  to  the  diagnosis 
of  the  nature  of  an  unknown  serum  or  other  solution  of  proteid. 
The  mere  fact  of  not  obtaining  a  precipitate  when  a  solution  of 
unknown  strength  (e.g.,  of  a  dried  blood-stain)  is  added  to  an 
anti-serum  is  not  necessarily  of  any  importance,  and  to  obtain 
accurate  results  it  is  necessary  to  perform  a  series  of  quantitative 
experiments. 

The  experiments  of  Eisenberg,  Michaelis,  Fleischmann,  and 
others  tend  to  show  that  the  combination  between  the  two  obeys 
the  laws  governing  the  combination  of  weak  acids  and  bases, 
which  we  have  already  discussed,  and  that  when  the  two  sera  are 
added  together  both  free  precipitin  and  precipitable  substance  may 
be  present  at  the  same  time.  This,  if  true,  would  not  explain 
relations  between  the  precipitin  and  precipitable  substance  similar 
to  those  given  above  ;  if  the  law  of  the  mass  reaction  applied, 
an  excess  of  antiserum  would  tend  to  give  the  maximum 
possible  quantity  of  precipitum,  though  there  would  always  be 
some  unaltered  precipitable  substance  present  in  an  unaltered 
state.  It  is,  however,  doubtful  whether  the  substances  do  actually 
interact  as  weak  acids  and  bases.  Von  Dungern,  experimenting 
with  antisera  obtained  to  the  proteids  of  cold-blooded  animals, 
found  that  the  reaction  was  rigorously  quantitative,  but  that  a 
complication  was  introduced  by  the  presence  of  two  varieties  of 
precipitin,  special  and  partial.  The  special  precipitins  are  sup- 


THE    PRECIPITINS  23! 

posed  only  to  act  on  the  proteid  which  has  been  used  for  the 
immunization,  whereas  the  partial  ones  act  both  on  it  and  on 
other  foreign  proteids.  If  these  are  formed  at  different  rates  of 
speed,  it  is  easy  to  see  that  at  a  certain  stage  in  the  process  of 
immunization  an  animal's  serum  may  contain  a  precipitin  (e.g., 
the  special  precipitin)  and  a  precipitable  substance  (e.g.,  that  which 
acts  as  an  antigen  to  the  partial  precipitin).  In  most  of  their 
reactions  the  precipitins  certainly  act  as  if  they  had  a  powerful 
combining  affinity  for  their  antigens,  and  von  Dungern's  observa- 
tions may  supply  the  explanation  of  the  apparent  discrepancies. 

If  precipitable  substance  (e.g.,  egg-albumin)  be  heated,  it  under- 
goes a  change  comparable  to  that  sustained  by  precipitin  on 
heating  :  it  loses  its  power  of  becoming  precipitated  with  anti- 
serum,  but  retains  its  function  of  combining  therewith.  This 
altered  proteid  is  sometimes  called  precipitoid,  since  it  is  analogous 
with  the  precipitoid  derived  from  precipitin  by  the  action  of  heat. 
The  term,  however,  is  a  bad  one,  since  its  use  leads  to  confusion 
between  the  two  substances,  and  the  only  word  which  is  at  all 
suitable  is  "precipitogenoid."  Precipitogenoid,  therefore,  consists 
of  the  haptophore  portion  of  the  molecule  of  precipitogen,  another 
portion  of  this  molecule  which  must  be  present  in  order  that  the 
reaction  may  take  place  being  destroyed.  From  the  fact  that  it 
retains  its  haptophore  group  we  should  expect  it  to  act  as  an 
antigen,  just  as  toxoid  does  ;  and  this  is  the  case,  for  the  injection 
of  precipitogenoid  calls  forth  the  production  of  ordinary  precipitin. 
In  any  complete  quantitative  study  of  the  interactions  of  serum 
and  antiserum  it  is  necessary  to  investigate  the  question  of  the 
presence  of  precipitogenoid  as  well  as  of  precipitoid,  and  the 
problems  thus  may  become  complicated  in  the  extreme. 

Precipitins,  like  the  agglutinins,  act  most  quickly  at  body 
temperatures,  and  show  their  essential  similarity  in  the  fact  that 
salts  are  necessary  for  both  reactions  ;  short  of  absolute  absence, 
the  amount  of  the  precipitum  form  depends  on  the  quantity  of 
salts  present  (Friedmann). 

There  is  an  interesting  analogy  between  the  agglutinins  and  the 
precipitins,  in  that  the  latter  as  well  as  the  former  are  occasionally 
seen  in  the  serum  of  unimmunized  animals,  though  the  precipitins 
are  much  more  uncommon  in  this  situation.  Thus,  Hoke  found 
the  presence  of  bacterio-precipitins  to  nitrates  from  B.  typhosus 
and  V.  cholera  fairly  frequently  present  in  the  serum  of  the  ox, 
more  rarely  in  that  of  the  goat,  pig,  and  sheep.  The  presence  of 


232  SPECIFICITY 

serum  precipitins  is  rarer,  but  occurs,  according  to  Noguchi,  in 
cold-blooded  animals  (crustacea,  etc.),  and  Lamb  found  in  normal 
rabbit  serum  a  precipitin  for  cobra  venom,  and  Obermayer  and 
Pick  one  for  dysglobulin  of  egg-white  in  the  same  fluid. 
N^The  relation  of  the  precipitins  as  regards  specificity  forms  a 
difficult  and  most  important  question.  It  was  at  first  thought 
that  the  specificity  was  a  sharp  one,  and  that  a  serum  prepared  by 
the  injection  of  human  serum  would  only  precipitate  with  human 
serum,  and  a  lacto-serum  prepared  by  the  injection  of  cow's  milk 
would  only  precipitate  with  that  fluid,  and  not  with  the  milk  of 
other  animals.  Thus,  Uhlenhuth  prepared  a  precipitin  by  inject- 
ing a  rabbit  with  ox  serum,  and  found  it  gave  a  precipitate  with 
that  fluid,  but  not  with  the  serum  of  the  horse,  donkey,  pig,  sheep, 
dog,  cat,  deer,  fallow-deer,  hare,  guinea-pig,  rat,  mouse,  rabbit, 
chicken,  goose,  turkey,  or  pigeon ;  but  he  also  showed  that  anti- 
egg  serum  was  not  sharply  specific,  and  would  coagulate  solutions 
of  albumin  from  eggs  other  than  those  of  the  species  used  for  the 
injection.  Wassermann  and  Stern  also  showed  that  antihuman 
serum  would  react,  though  but  slightly,  with  the  serum  of  the 
baboon,  and  Stern  confirmed  this,  and  found  it  reacted  with  the 
serum  of  other  monkeys.  Hence  the  idea  gradually  arose  that 
the  precipitin  obtained  by  the  injection  of  a  serum  from  one  species 
of  animal  is  not  specific  for  that  species,  but  will  give  a  precipi- 
tate, though  of  less  amount,  with  the  sera  of  other  species,  provided 
that  they  are  sufficiently  closely  allied  zoologically.  This  has  been 
especially  studied  by  Nuttall,  who  expresses  this  relationship 
between  species  close  together  in  the  animal  scale  as  a  "  blood- 
relationship."  He  showed  that,  provided  the  serum  were  powerful 
enough,  it  would  react  with  all  the  bloods  of  animals  in  the  same 
great  division  of  the  animal  kingdom  (mammalia,  birds,  reptiles, 
amphibia,  etc.).  Thus,  a  strong  antihuman  serum  will  give  a 
precipitate  with  human  serum,  even  when  highly  diluted,  with 
apes,  monkeys,  etc.,  but  not  in  such  high  dilutions,  and  a  slight 
trace  of  precipitate  after  a  long  period  when  mixed  with  the  sera 
of  more  remote  mammalia,  but  no  precipitate  with  the  blood  of 
birds,  fishes,  etc.  Quite  similar  relationships  hold  with  the 
lacto-sera  and  with  the  precipitating  sera  for  muscle  proteids; 
the  anti-sera  for  egg  proteids  is  apparently  less  specific. 

Hence  we  deduce  that  the  precipitins  are  not  specific  as  regards 
the  animal  species  from  which  they  are  derived,  but  possess  that 
partial  specificity  seen  in  the  cytotoxins  and  in  the  "  group 


THE    PRECIPITINS  233 

reactions  "  of  the  agglutinins  ;  that  is  to  say,  they  are  specific  as 
regards  the  antigens  which  bring  them  into  existence,  irrespective 
of  the  source  from  which  that  antigen  was  derived.  This  appears 
to  be  substantiated  by  experiments  on  purified  and  recrystallized 
proteids,  the  precipitating  sera  for  which  show  a  high  degree  of 
specificity.  Too  frequent  recrystallization  of  proteids,  however, 
appears  to  injure  their  power  of  inducing  the  formation  of 
precipitins. 

Further,  proteids  which  have  been  altered  in  character  by 
chemical  processes  (iodized,  nitrified,  or  denitrified)  will  cause 
the  formation  of  precipitins  which  are  specific  for  the  transformed 
molecules  of  proteid,  no  matter  from  what  source  they  were 
derived.  These  observations  are  of  profound  interest,  since  they 
appear  to  show  that  it  is  possible  by  artificial  means  to  alter  com- 
pletely the  haptophore  group  of  a  proteid  molecule.  According 
to  Uhlenhuth,  there  is  little  or  no  specificity  in  the  antisera  pre- 
pared against  the  proteids  of  the  crystalline  lens.  An  antiserum 
prepared  by  injections  of  serum  will  precipitate  all  albuminous 
fluids  except  a  solution  of  the  lens,  which  in  its  turn  will  not  pre- 
cipitate with  serum.  But  an  anticrystalline  serum  prepared  by 
the  injection  of  ox  lenses  into  rabbits  will  give  precipitates  with 
lens  solutions  from  mammals,  birds,  amphibia,  and  even  fish. 

Hence,  too,  a  practical  point  in  connection  with  the  employment 
of  precipitating  sera  in  the  detection  of  the  source  of  blood.  Here 
it  is  necessary  to  use  a  high  dilution  of  the  blood  to  be  tested,  and 
if  a  reaction  is  given,  to  try  again  with  higher  dilutions  until  the 
limit  is  reached.  In  testing  dried  blood-stains  for  medico-legal 
purposes  it  is,  of  course,  usually  impossible  to  determine  the  exact 
amount  of  serum  which  has  been  taken  up  by  the  solvent  (normal 
saline  solution).  It  is  found,  however,  that  a  i  :  1,000  dilution 
of  serum  in  normal  saline  solution  will  give  a  good  froth  when  air 
is  allowed  to  bubble  through  them  from  a  pipette,  and  this  will 
give  a  rough  idea  as  to  the  amount  of  proteid  material  dissolved 
out  of  the  clot  to  be  tested. 

The  delicacy  of  the  reaction  is  truly  astonishing.  Thus  Ascoli 
obtained  an  anti-egg  albumin  serum  which  gave  a  precipitate  with 
a  i  :  1,000,000  dilution  of  egg-albumin,  and  Stern  an  antihuman 
serum  which  reacted  with  serum  at  a  dilution  of  i  :  50,000.  These 
are  extreme  figures,  but  sera  active  on  solutions  diluted  i  :  5,000  are 
frequently  obtained. 

As  regards  the  substances  for  which  precipitins  can  be  obtained, 


234  ISOPRECIPITIN 

we  find  them  limited  in  all  cases  to  the  proteids.  All  the  coagu- 
lable  proteids  will  give  rise  to  the  formation  of  precipitating  sera. 
As  regards  the  formation  of  these  substances  by  means  of  digested 
proteids  (peptone  and  albumoses),  the  facts  are  less  certain. 
Myers  obtained  a  precipitin  to  Witte's  peptone,  but  according  to 
Obermayer  and  Pick  complete  peptic  digestion  destroys  both  the 
power  of  inducing  the  formation  of  antibodies  and  of  being  pre- 
cipitated. The  former  is  first  to  go,  so  that  a  partially  digested 
proteid  solution  which  will  no  longer  precipitate  with  an  antiserum 
will  yet  lead  to  the  production  of  a  precipitin  when  injected  into  a 
suitable  animal.  The  results  with  tryptic  digestion  appear  to  be 
entirely  similar.  Complete  destruction  leads  to  loss  of  both 
functions.  It  appears,  therefore,  that  it  is  only  the  giant  molecule 
of  proteid  that  can  be  agglutinated  ;  the  smaller,  diffusible 
molecule  of  the  products  of  proteolysis,  like  the  molecules  of 
toxin,  though  they  may,  and  probably  do,  unite  with  their  anti- 
bodies, do  not  manifest  this  combination  by  forming  clumps. 
Proteids  which  have  been  coagulated  by  heat  have  still  the  power 
of  forming  a  precipitin  (which,  of  course,  manifests  its  action  on 
solutions  of  the  unaltered  proteid)  when  injected. 

As  regards  the  species  of  animal  from  which  the  precipitins  are 
to  be  prepared,  it  is  natural  to  choose  an  animal  as  remote  in  the 
zoological  scale  as  possible.  In  the  case  of  human  sera  or  in- 
different substances,  such  as  solutions  of  albumin,  etc.,  the  rabbit 
is  usually  chosen,  since  it  is  easily  obtained  and  handled,  and  will 
yield  a  very  considerable  amount  of  serum.  Where  antisera  to 
animals  closely  allied  to  rabbits  are  to  be  obtained,  fowls  or  other 
large  birds  are  most  suitable.  There  are,  however,  some  observa- 
tions which  go  to  show  that  there  are  differences  between  individual 
rabbits  which  are  comparable  in  every  respect  to  those  between 
the  different  red  corpuscles  of  goats  and  other  animals,  as  seen  in 
Ehrlich's  experiments  on  the  isohaemolysins.  Thus  Schiitze 
injected  the  serum  of  rabbits  into  rabbits.  In  two  cases  out  of 
ten  he  obtained  a  precipitin  which  reacted  with  the  serum  injected. 
This  substance  is  called  isoprecipitin.  It  may  often  be  noted 
that  a  precipitate  falls  when  different  samples  of  diphtheria  anti- 
toxin (in  animals  in  which  the  earlier  stages  of  the  process  of 
immunization  have  been  carried  out  by  means  of  serum  toxin)  are 
mixed  together,  and  this  is  probably  an  analogous  phenomenon. 
These  precipitins  are,  however,  very  feeble  as  compared  with 
those  obtained  by  the  injection  of  the  serum  of  a  certain  species 


THE    PRECIPITINS  235 

into  an  animal  far  removed  in  the  zoological  scale,  and  their 
explanation  is  obvious  in  the  light  of  what  has  been  already  said 
with  regard  to  individual  differences  in  receptors. 

According  to  Ewing,  an  antihuman  serum  prepared  from  the 
fowl  shows  a  far  higher  degree  of  specificity  than  those  obtained 
by  injections  into  rabbits. 

A  short  account  of  the  practical  application  of  the  precipitins 
may  not  be  out  of  place.  The  chief  is,  of  course,  the  medico-legal 
identification  of  blood-stains,  the  chief  exponents  of  which  are 
Uhlenhuth  and  Wassermann.  The  antiserum  is  obtained  from 
rabbits,  which  are  treated  by  intravenous  or  intraperitoneal  in- 
jections at  intervals  of  three  or  four  days.  The  material  used 
for  the  injection  may  be  blood  obtained  by  venesection  or  vein 
puncture,  or  from  the  placenta  or  from  the  cadaver,  or  pleuritic  or 
ascitic  fluid  may  be  used ;  in  any  case  strict  asepsis  is  necessary. 
The  amount  given  rises  from  i  to  3  or  4  c.c.  in  the  case  of 
intravenous  injections,  or  twice  as  much  or  even  more  in  the 
peritoneum.  The  course  of  treatment  lasts  three  or  four  months. 
Another  and  simpler  method  is  to  give  larger  doses — up  to  10  c.c., 
or  even  more — intraperitoneally  at  intervals  of  a  week.  The 
animal  is  then  chloroformed,  and  as  much  blood  as  possible 
collected  either  from  the  heart  or  carotid  artery. 

The  fluid  to  be  tested  is  prepared  by  maceration  of  the  clot, 
piece  of  stained  linen,  etc.,  with  normal  saline  solution,  or  with 
i  per  cent.  NaOH.  In  the  case  of  a  very  old  stain,  Ziemka 
recommends  the  use  of  a  strong  solution  of  potassium  cyanide, 
which  is  subsequently  neutralized  with  tartaric  acid.  This  is 
examined  with  the  microscope  and  tested  spectroscopically  to 
determine  the  presence  of  blood-corpuscles  and  pigments.  The 
solution  is  then  filtered.  Air  is  then  allowed  to  bubble  through 
the  fluid  to  make  sure  that  proteid  material  has  actually  passed 
into  solution ;  this  is  indicated  by  the  production  of  a  stable  foam. 
Three  tests  are  made.  In  the  first  tube  one  part  of  the  fluid  under 
examination  is  mixed  with  two  of  the  antiserum,  the  second 
contains  the  fluid  alone,  and  the  third  antiserum  plus  normal 
saline  solution.  Further  controls,  in  which  the  antiserum  is 
mixed  with  diluted  serum  from  animals  other  than  man,  may  also 
be  made  if  necessary.  The  tubes  are  usually  incubated  and 
examined  from  time  to  time,  and  a  positive  result  is  obtained  if 
there  is  a  precipitate  in  the  first  tube  and  not  in  the  others. 
Further  tests  are  then  made  with  greater  dilutions,  and  with  a 


236  DEVIATION    OF   COMPLEMENT   TEST 

powerful  antiserum  a  reaction  can  usually  be  obtained  in  dilutions 
so  high  that  proof  of  the  presence  of  proteids  is  barely  obtainable 
by  ordinary  chemical  means.  The  weak  point  in  the  method  is 
that  it  is  never  possible  to  say  exactly  how  much  of  the  proteid 
matter  of  the  clot  has  been  dissolved,  and  thus  to  compare  the 
effect  of  the  antiserum  on  the  solution  with  its  action  on  diluted 
serum  of  man  and  of  other  animals.  Given  a  sample  of  unaltered 
and  undried  serum,  the  test  can  be  carried  out  with  almost 
complete  certainty ;  but  this  is  rarely,  if  ever,  possible  in  actual 
practice. 

Another  test,  based  on  Gengou's  reaction,  has  recently  been  intro- 
duced by  Neisser  and  Sachs.  It  is  carried  out  in  the  following 
way  :  A  hsemolysin  for  ox  corpuscles  is  prepared  by  injecting 
these  bodies  into  a  rabbit.  Another  rabbit  is  injected  with  human 
blood,  so  as  to  lead  to  the  production  of  a  precipitin.  When  the 
test  is  to  be  made  the  fresh  serum  of  the  latter  animal  (or  if  only 
stale  serum  is  at  hand,  some  fresh  normal  rabbit's  serum  must  be 
added  to  supply  complement)  is  added  to  the  fluid  to  be  tested. 
If  human  serum  is  present,  even  in  an  amount  so  small  that  no 
precipitate  is  formed,  the  antigen  and  antibody  combine  and  with- 
draw the  complement  from  solution.  To  test  this  ox  corpuscles 
are  sensitized  with  the  heated  (decomplemented)  serum  of  the  first 
rabbit  and  thoroughly  washed,  and  some  of  the  mixture  added. 
If  the  complement  has  been  withdrawn,  of  course  no  haemolysis 
will  occur.  Certain  obvious  controls  are  employed  to  demonstrate 
that  the  corpuscles  were  actually  sensitized,  and  that  complement 
was  present  in  the  rabbit's  serum  before  the  addition  of  the  fluid 
suspected  of  containing  human  blood. 

This  test  is  extraordinarily  sensitive,  Neisser  and  Sachs  finding 
that  the  millionth  part  of  a  cubic  centimetre  of  human  serum  was 
readily  demonstrable.  They  claim  also  that  it  is  more  specific 
than  the  ordinary  precipitin  test,  it  being  necessary,  for  instance, 
to  use  T^Viy  c.c.  of  ape's  serum  to  get  the  same  result.  But  the 
technique  is  complicated,  and  it  appears,  moreover,  that  comple- 
ment may  be  abstracted  in  an  altogether  non-specific  manner  by 
substances  other  than  the  combination  of  antigen  and  antibody. 
Thus  Uhlenhuth  examined  some  spots  of  blood  on  a  sack  which  a 
cabdriver  (who  was  found  dead)  used  as  a  seat,  and  found  it 
brought  about  a  fixation  of  complement,  though  it  gave  no  reaction 
with  the  precipitin  test.  He  then  tested  the  material  of  which 
the  bag  was  made,  and  found  it  also  had  the  power  of  absorbing 


THE    PRECIPITINS 


237 


complement.  According  to  Neisser  and  Sachs,  however,  this 
power  is  destroyed  if  the  serum  solution  is  boiled,  but  is  unaltered 
in  the  case  of  non-specific  substances.  Another  serious  objection 
is  that  a  similar  deviation  may  be  brought  about  by  means  of 
sweat,  so  that  if  the  reaction  were  obtained  in  a  stain  on  body- 
linen  it  would  be  of  little  value. 

The  precipitin  reaction  has  also  been  used  for  determining  the 
nature  of  meat  (whether  fresh,  as  in  the  case  of  beef  suspected  to 
be  horse-flesh,  or  prepared,  as  in  sausages,  etc.).  The  serum  is 
prepared  by  injecting  meat-juice  or  an  (unheated)  watery  extract 
of  the  meat,  and  the  test  is  carried  out  on  lines  similar  to  those 
described  above.  It  has  also  been  employed  to  determine  the 
nature  of  bones,  i.e.,  whether  they  are  human  or  otherwise. 


CHAPTER  X 
PHAGOCYTOSIS 

METCHNIKOFF'S  researches  on  phagocytosis  in  the  lowly  organized 
animals  formed  a  starting-point  for  an  entirely  new  series  of 
researches  on  the  subject  of  immunity,  and  his  treatise  on  the 
{<  Comparative  Pathology  of  Inflammation  "  must  ever  remain  a 
great  medical  classic,  as  well  as  a  most  fascinating  work. 
Metchnikoff  was  primarily  a  biologist,  and  his  attention  was 
attracted  to  the  spectacle  of  amoebae  and  other  unicellular 
organisms  containing  bacteria.  In  these  lowly  constituted 
animals  the  process  by  which  the  cell  takes  in  foreign  particles 
can  be  watched  with  ease,  and  the  steps  of  the  process  traced. 
The  amoeba  throws  out  arm-like  processes,  which  surround  the 
bacterium,  close  on  it,  and  in  a  few  minutes  an  organism 
which  was  previously  lying  free  is  deeply  embedded  in  the 
animal's  protoplasm.  Metchnikoff  watched  its  fate,  and  found 
it  lost  its  sharp  outline  and  clear  appearance,  became  granular, 
and  in  a  little  while  disappeared  altogether.  He  found  also  that, 
in  many  cases  at  least,  a  minute  vacuole  was  formed  round  the 
ingested  organism,  and  further  research  showed  that  this  vacuole 
contained  a  fluid  which  was  acid  in  reaction  and  held  in  solution 
a  digestive  ferment  allied  to  pepsin.  Considering  this  observation 
more  closely,  it  is  obvious,  firstly,  that  the  amoeba  must  be  regarded 
as  being  immune  to  the  organisms  which  it  ingests  and  digests, 
and,  secondly,  that  in  this  case  the  processes  concerned  in  im- 
munity are  those  concerned  in  nutrition  :  the  amoeba  is  immune 
to  the  bacterium  because  it  can  make  use  of  its  protoplasm  as 
nourishment. 

But  this  process,  as  Metchnikoff  soon  found,  is  not  confined 
to  the  unicellular  organisms.  His  most  beautiful  and  classical 
series  of  researches  deal  with  the  properties  and  action  of  the 
leucocytes  in  a  small  fresh-water  crustacean  (daphnia),  which, 
from  its  transparency  and  small  size,  is  a  very  suitable  object  for 

238 


PHAGOCYTOSIS  239 

observation.  Leucocytes,  it  may  be  pointed  out,  are  cells  which, 
from  their  isolation  from  one  another,  power  of  independent  move- 
ment, etc.,  are  closely  analogous  with  amoebae  and  other  lowly 
organized  protozoa. 

Metchnikoff  found  that  daphnia  is  subject  to  a  disease  due  to 
the  invasion  of  its  body-cavity  by  the  spores  of  a  yeast— the 
monospora — and  that  if  these  spores  gained  access  in  large 
numbers  they  multiplied,  formed  into  mature  organisms,  and 
finally  killed  their  host.  When,  however,  few  spores  gained 
access,  he  found  that  the  daphnia's  leucocytes  approached  them, 


FIG.  46.— AN  AMCEBA  WHICH  HAS  INGESTED  NUMEROUS  SPECIMENS  OF 
MICROSPH^RA.    (Metchnikoff.) 

a,  #,  Vacuoles. 

formed  a  wall  round  them,  and  finally  digested  and  destroyed 
them.  It  is  obvious,  therefore,  that  the  immunity  of  these 
animals  is  relegated,  so  to  speak,  to  its  leucocytes.  If  these  are 
efficient,  the  animal  is  preserved  from  its  invaders;  whereas, 
if  they  make  default,  the  latter  multiply,  and  bring  about  the 
lethal  issue. 

MetchnikofFs  experiments  were  by  no  means  confined  to  the 
action  of  the  leucocytes  on  bacteria.  They  included  a  careful 
and  exhaustive  study  of  the  method  of  absorption  of  all  manner 
of  particles,  organized  and  unorganized,  in  the  tissues  and  body- 
cavities  of  animals  of  all  positions  in  the  animal  world,  and  they 
proved  to  the  full  the  importance  of  phagocytosis  and  intra- 
cellular  digestion,  and  one  of  the  chief — if  not,  indeed,  the  only — 


240 


PHAGOCYTOSIS    IN    THE    LOWER    ANIMALS 


method  by  which  intruding  particles  are  dealt  with  in  the  animal 
economy.  And,  further,  they  correlate  in  the  clearest  possible 
way  this  "  scavenging  "  of  the  tissues  with  normal  processes  of 


FIG.  47. — DAPHNIA  CONTAINING  LARGE  NUMBERS  OF  MONOSPOR/E. 
(Metchnikoff.) 


FIG.  48. — HIND  PART  OF  DAPHNIA. 
At  a  the  spores  of  monospora  are  shown  surrounded  by  leucocytes. 

digestion  and  nutrition.  Take,  for  instance,  the  absorption  of 
the  red  blood-corpuscles  of  birds,  which  are  very  suitable  for 
research,  being  readily  recognized  (they  are  nucleated),  non- 
poisonous,  and  easily  digested.  Metchnikoff  showed  that  these 


PHAGOCYTOSIS  241 

substances  are  absorbed  from  the  alimentary  canal  of  the  lower 
invertebrates  by  a  process  of  intracellular  digestion,  and  by  that 
alone.  Thus,  when  the  alimentary  canal  of  a  planarian  (Dendro- 
ccelmn  lacteum,  an  animal  resembling  the  liver-fluke  in  its  general 
anatomy)  is  filled  with  blood,  the  latter  is  found  to  undergo  the 
changes  in  colour  familiar  in  bruises,  and  this  is  due  to  the  fact 
that  the  blood  has  been  taken  from  the  alimentary  canal  by  the 
cells  lining  it,  and  there  has  undergone  the  digestive  changes. 
These  processes  can  readily  be  traced  under  the  microscope,  and 
the  blood-corpuscles  can  be  seen,  at  first  embedded  in  protoplasm 
and  of  normal  contour,  and  later  enclosed  in  vacuoles  and  of  altered 
shape.  Complete  digestion  takes  several  days,  and  every  stage  of 
the  process  is  easy  to  watch.  Absorption  of  organized  food  particles 
takes  place  from  the  alimentary  canal  in  exactly  the  same  way 
in  actinians,  molluscs,  and  many  other  lower  animals,  and  in 
many  of  the  cases  which  have  been  investigated  the  vacuoles  are 
found  to  contain  a  digestive  ferment  allied  to  trypsin  or  pepsin. 
In  higher  animals  this  process  of  intracellular  digestion  does  not 
occur,  or  only  to  a  slight  extent  (in  the  case  of  fats),  the  animal 
finding  it  more  advantageous  to  secrete  the  digestive  juices  into 
the  alimentary  canal,  and  to  absorb  the  products  of  their  action 
therefrom  in  a  state  of  solution. 

Absorption  of  particulate  substances  which  have  gained  access 
to  the  tissues  takes  place,  to  a  very  large  extent,  in  a  method 
entirely  similar.  Thus  Metchnikoff  showed  that  when  bird's 
corpuscles  are  injected  into  the  tissues  of  the  larva  of  the  cock- 
chafer, or  into  the  snail,  earthworm,  the  peritoneal  cavity  of  the 
goldfish,  etc.,  the  process  is  also  intracellular  and  entirely  similar 
to  those  which  occur  when  these  corpuscles  are  injected  into  the 
alimentary  canal  of  the  planarian.  In  most  cases  the  corpuscles 
are  absorbed  by  the  leucocytes,  in  others  by  the  cells  of  the  part 
(such  as  the  endothelial  cells  lining  the  peritoneum) ;  but  in  all 
cases  there  is  the  same  vacuolation,  the  same  series  of  changes 
in  the  ingested  corpuscles,  and  the  same  final  result.  We  shall 
not  be  far  wrong  in  associating  the  absorption  of  these  corpuscles 
in  a  very  close  way  with  processes  of  digestion  and  nutrition. 

The  French  school  have  studied  these  processes  of  absorption 
of  particulate  bodies  from  the  tissues  in  a  very  complete  manner, 
and  have  shown  beyond  dispute  the  importance  of  phagocytosis 
in  this  respect.  Thus  particles  of  carbon  in  the  lung,  the  granules 
of  pigment  left  after  interstitial  haemorrhages,  etc.,  are  all  ingested 

16 


2^2.  METCHNIKOFF'S  THEORY  OF  IMMUNITY 

and  removed  by  phagocytes  of  one  sort  or  another,  and  this  dis- 
covery throws  a  flood  of  light  on  the  meaning  of  many  of  the 
phenomena  of  inflammation,  more  especially  on  the  leucocytic 
invasion  of  the  injured  tissues,  which  has  for  its  object,  in  part 
at  least,  the  removal  of  particulate  substances  which  are  the  cause 
of  the  injury.  Further,  the  dead  or  injured  tissues,  the  result  of 
the  action  of  the  irritant,  are  eroded  and  removed  by  phagocytic 
action.  If  a  small  volume  of  tissue,  situated  in  a  region  to  which 
numerous  leucocytes  can  gain  access,  be  killed,  it  may  be  com- 
pletely removed  piecemeal  in  this  way.  We  may  quote  as  an 
example  the  complete  absorption  of  the  central  slough  which 
often  takes  place  in  acne  pustules  or  small  boils,  especially  after 
treatment  with  staphylococcic  vaccine.  Very  interesting  in  this 
connection  is  the  process  of  the  absorption  of  the  tail  of  the 
tadpole,  which  is  removed  in  an  entirely  similar  way  by  the  action 
of  phagocytes.  And  it  is  worthy  of  notice  that  the  digestive 
power  of  the  phagocytes  is  a  very  powerful  one,  and  substances 
usually  deemed  entirely  insoluble  may  be  gradually  removed  by 
their  action. 

We  have  already  referred  to  the  action  of  leucocytes  in  ab- 
sorbing and  neutralizing  toxins,  and  have  quoted  the  beautiful 
experiments  of  Besredka  on  trisulphide  of  arsenic,  which,  when 
placed  'in  the  peritoneal  cavity,  is  absorbed  by  these  cells  and 
prevented  from  exercising  its  poisonous  action ;  whilst,  when 
shielded  from  their  attack  by  being  enclosed  in  bags  which  are 
permeable  to  fluids,  but  not  to  solids,  it  undergoes  gradual 
solution,  and  leads  to  fatal  intoxication. 

Metchnikoff  applied  these  researches  on  phagocytosis  to  the 
question  of  immunity,  and  formulated  a  complete  and  logical 
theory  on  the  subject,  which  he  illustrated  with  many  striking 
researches  and  examples.  For  him — and  modern  advances  tend 
more  and  more  to  corroborate  the  truth  of  this  view,  though  in  a 
much  more  complicated  way  than  he  thought — the  defence  of  the 
animal  economy  is  entrusted  entirely  to  the  phagocytes,  and 
especially  to  the  leucocytes.  If  a  bacterium  enters  the  tissues 
these  cells  may  at  once  make  their  way  to  the  seat  of  infection, 
and  proceed  to  ingest  the  bacteria  and  kill  them  intracellularly. 
In  this  case  the  animal  recovers,  with  or  without  a  transient  illness, 
and  we  say  it  is  immune.  If  another  species  of  bacterium  enters, 
the  effects  may  be  different :  the  leucocytes  may.  perhaps,  be 
repelled  instead  of  being  attracted,  or,  if  attracted,  may  be  killed 


PHAGOCYTOSIS  243 

by  the  action  of  the  bacterial  toxins,  so  that  no  phagocytosis 
occurs  ;  or  perhaps  they  may  take  up  the  bacteria  and  then  be 
killed  by  the  toxins.  In  any  case,  the  result  is  the  same  :  the 
bacteria  continue  to  grow  and  to  produce  their  toxins,  and  the 
result  is  death.  The  animal  is  susceptible  to  the  bacterium 
because  its  leucocytes  are  unable  to  deal  with  it.  Immunity, 
therefore,  is  a  function  of  the  phagocytes. 

So  far  we  have  dealt  with  natural  immunity.  The  application 
of  Metchnikoff's  theory  to  acquired  immunity  is  equally  simple, 
though  much  less  satisfactory.  He  argues  that  the  leucocytes, 
in  their  contest  with  a  particular  species  of  bacterium,  become 
educated  to  overcome  this  bacterium,  and  are  able  to  deal  with  it 
in  future  with  great  ease.  In  the  first  infection  there  may  be  a 
balanced  contest  of  some  severity  and  duration,  but  as  a  result 
the  leucocytes,  like  war-trained  veterans,  are  readily  able  to  cope 
with  the  invader  a  second  time.  This  theory,  though  ingenious, 
cannot  be  maintained  at  the  present  day.  Its  truth  rests,  of 
course,  on  the  truth  of  Metchnikoff's  main  thesis,  which  is  only 
partially  true,  and  which  is  only  one  factor  in  the  complicated 
phenomena  of  immunity.  We  may  just  point  out,  however,  that 
the  life  of  a  leucocyte  is,  in  all  probability,  a  comparatively  short 
one,  to  be  measured  by  days,  or  at  most  weeks,  so  that  acquired 
immunity  due  to  the  education  of  the  leucocytes  would  be  of 
short  duration.  Nor  is  it  of  any  assistance  to  argue  that  in  the 
struggle  against  the  invading  bacterium  the  fittest  leucocytes 
would  survive,  and  so  lead  to  the  general  improvement  of  the 
leucocyte  species :  for  leucocytes  do  not  propagate  themselves, 
but  are  emitted  from  the  bone-marrow,  run  their  course  in  the 
blood,  degenerate,  and  die,  their  place  being  taken  by  others  from 
the  same  source.  The  education,  therefore,  must  be  one  of  the 
bone-marrow,  and  we  cannot  conceive  how  this  could  take  place 
as  the  result  of  phagocytosis  going  on  in  a  distant  area.  It  is 
possible  that  something  of  the  sort  may  occur,  but  only  by  the 
action  of  toxins  and  other  bacterial  products  circulating  in  the 
blood-stream,  and  being  thus  brought  into  the  marrow.  Other 
considerations  might  be  urged,  but  the  theory  has  now  but  a 
historic  interest.  It  has  served  its  purpose :  it  has  been  the 
means  of  suggesting  many  researches  which  have  helped  greatly 
in  the  elucidation  of  a  most  difficult  subject. 

Before  discussing  the  role  of  phagocytosis  in  the  infective  pro- 
cesses we  must  deal  briefly  with  two  subjects  :  firstly,  the  means 

1 6 — 2 


244  CHEMOTAXIS 

by  which  the  phagocytes  are  brought  into  contact  with  the 
bacteria — in  other  words,  of  chemotaxis.  This  is  a  phenomenon 
which  is  displayed  by  almost  all  motile  and  unicellular  organisms, 
whether  animal  or  vegetable,  and  by  the  leucocytes  of  the  higher 
animals,  and  manifests  itself  in  a  movement  of  the  organism  in 
response  to  a  chemical  stimulus.  To  take  an  example  from  the 
bacteria  :  if  a  capillary  tube  containing  a  solution  of  meat-extract 
be  placed  in  a  watery  emulsion  of  B.  coli  or  B.  typhosus,  the 
bacteria  will  be  seen  to  group  themselves  round  the  mouth  of  the 
tube,  which  they  ultimately  enter.  This  is  an  example  of  positive 
chemotaxis,  the  bacteria  being  attracted  by  the  extractives,  which, 
indeed,  they  utilize  as  food.  On  the  other  hand,  bacteria  will 
tend  to  remove  themselves  from  an  area  from  which  alcohol  and 
/  similar  substances  are  diffusing  :  this  is  negative  chemotaxis.  As 
a  general  rule,  we  may  say  that  motile  organisms  tend  to  be 
attracted  into  a  region  rich  in  useful  and  nutritious  substances, 
and  are  repelled  from  injurious  ones,  but  this  is  not  invariably 
true  in  the  artificial  conditions  of  experiment.  An  organism  may 
be  lured  by  a  useful  substance  into  a  region  where  there  is  a 
sufficient  quantity  of  an  injurious  one  to  kill  it. 

In  cases  where  leucocytes  make  their  way  into  a  tissue  infected 
with  bacteria  it  must  be,  therefore,  because  the  latter  give  off  a 
substance  which  has  a  positive  chemotactic  action  on  them.  This 
action  may  readily  be  shown  by  experiment,  and  the  easiest  way 
is  to  work  with  the  lymph  of  a  cold-blooded  animal,  since  in  this 
way  all  difficulties  connected  with  a  warm  stage  are  avoided.  If, 
for  instance,  we  take  a  few  anthrax  bacilli  and  place  them  on  a 
slide,  add  a  drop  of  frog's  lymph  (from  the  dorsal  lymph  sac),  and 
apply  a  cover-glass,  the  leucocytes  can  be  easily  seen  to  crawl 
actively  up  to  the  bacilli.  To  this  experiment  (Kanthack's)  we 
shall  have  to  recur. 

In  nearly  all  cases  we  find  that  leucocytes  are  attracted  in  large 
numbers  into  the  area  in  which  the  bacteria  are  situated — i.e.. 
nearly  all  bacteria  give  off  substances  which  are  positively 
chemotactic  for  leucocytes.  In  a  few  cases,  however,  this  appears 
at  first  sight  not  to  happen,  for  when  tissues  which  are  infected 
with  very  virulent  bacteria  are  examined  microscopically,  they 
are  often  extremely  poor  in  leucocytes.  As  an  example,  we  may 
take  any  example  of  acute  spreading  gangrene  of  bacterial  origin. 
But,  as  Kanthack  pointed  out,  these  are  not  necessarily  examples 
of  negative  chemotaxis,  and  it  is  quite  probable  that  the  paucity 


PHAGOCYTOSIS  245 

of  the  leucocytes  is  due  to  their  paralysis  and  destruction  by  the 
powerful  toxins  which  are  given  off.  Certainly  since  the  use  of 
methods  involving  experiments  on  phagocytosis  in  vitro  no  valid 
example  of  negative  chemotaxis  has  been  adduced,  and  it  is  highly 
probable  that  leucocytes  are  attracted  by  all  bacteria,  whether 
their  toxins  are  mild  or  potent.  In  the  former  case  the  leucocytic 
infiltration  will  be  very  obvious ;  in  the  latter  the  cells  will  be  first 
paralyzed  and  then  destroyed  as  soon  as  they  reach  an  area  in 
which  the  toxin  is  present  in  a  high  degree  of  concentration. 

We  must  not  assume  that  this  property  of  being  attracted  by 
bacteria  is  of  any  advantage  to  the  leucocyte  itself,  arguing  from 
the  fact  that  the  free-swimming  unicellular  organisms  are  attracted 
by  food  and  oxygen.  The  leucocytes  are  in  many  cases  attracted 
into  the  infected  area  to  their  own  undoing,  and  it  must  not  be 
forgotten  that  even  in  an  inflammatory  process  which  is  mild  in 
nature  and  favourable  in  result  the  number  of  leucocytes  which 
may  be  killed  in  the  conflict  is  enormous.  The  leucocytes  are  not 
independent  protozoa  inhabiting  the  blood  and  tissues,  but  an 
integral  part  of  the  organism.  It  is  to  the  advantage  of  the  latter 
that  the  former  should  be  attracted  at  once  to  the  seat  of  invasion, 
and  hence  the  processes  of  evolution  have  led  to  the  development 
of  this  function  in  the  nomadic  cells  of  the  body.  These  are 
extraordinarily  susceptible  to  chemotactic  influences  ;  they  seem 
to  be  attracted  by  any  deviation  from  the  normal  constitution  of 
the  tissues  and  fluid  :  a  slight  injury,  a  haemorrhage,  the  presence 
of  a  poison,  of  a  foreign  body  of  any  sort  or  of  any  dead  or  useless 
tissue,  and  the  leucocytes  are  immediately  attracted  into  the  area 
affected.  The  more  we  regard  the  process,  the  more  we  must 
regard  it  as  one  of  the  most  exquisite  examples  of  means  to  ends 
met  with  in  the  animal  economy. 

Secondly,  with  regard  to  the  nature  of  the  cells  which  have  the 
power  of  acting  as  phagocytes.  Of  these  the  most  important  are 
the  leucocytes,  and  especially  the  polynuclear  and  large  hyaline 
cells  of  human  blood.  All  the  leucocytes,  however,  have  phago- 
cytic  powers,  as  is  well  seen  in  opsonic  estimations :  the  cells 
which  take  up  the  fewest  bacteria  are  the  eosinophiles  and  the 
small  lymphocytes.  The  former  are  very  deficient  in  this  direc- 
tion, and  we  may  be  certain  that  their  main  function  is  entirely 
different.  The  lymphocytes,  too,  take  up  but  a  small  number  of 
bacteria  ;  but  when  activated  by  a  suitable  haemopsonic  serum,  they 
take  up  red  blood-corpuscles  in  considerable  numbers,  and  a 


246  VARIETIES    OF    PHAGOCYTES 

lymphocyte  may  then  present  a  remarkable  appearance,  having 
ingested  two  or  even  three  red  corpuscles,  each  as  large  as  itself, 
the  narrow  band  of  protoplasm  being  extended  over  the  corpuscle 
in  a  most  extraordinary  way. 

It  is  noticeable,  too,  that  even  among  the  polynuclears  there  are 
great  differences  in  phagocytic  powers.  This  is  best  seen  in  a  film 
of  pus  from  a  case  of  gonorrhoea,  in  which  certain  cells  (indis- 
tinguishable from  the  others)  are  packed  full  of  cocci,  whereas  the 
vast  majority  are  entirely  free.  The  same  fact  is  brought  out 


FIG.  49. — RED  CORPUSCLES  INGESTED  BY  POLYNUCLEAR  LEUCOCYTES 
AND  LYMPHOCYTES.     (Original.) 

clearly  in  opsonic  experiments,  where  some  leucocytes  are  often 
found  to  take  up  very  large  numbers  of  bacteria,  the  general 
average  of  the  other  cells  being  low. 

Besides  the  leucocytes,  some  of  the  tissue  cells,  which  are 
either  free  or  have  the  power  of  becoming  so,  are  active  phago- 
cytes. Of  these,  the  most  important  are  the  endothelial  cells. 
These  are  only  flat  plates  of  protoplasm  when  under  normal 
conditions.  When  submitted  to  the  action  of  almost  any  irritant 
they  become  cuboidal  or  columnar,  and  are  then  most  active 
phagocytes.  A  good  example  of  this  may  sometimes  be  seen  in 
sections  of  a  thrombosed  vein  at  a  certain  stage :  the  endothelial 
cells  are  columnar  and  contain  much  protoplasm,  and  this  latter 
is  packed  with  pigment  granules  absorbed  from  the  altered  blood 
in  the  lumen.  But  the  process  goes  farther  than  this,  and  the 
endothelial  cell  (whether  of  the  serous  membranes,  vessels,  or 
lymph  clefts)  either  breaks  loose  from  its  attachments  or  buds  off 


PHAGOCYTOSIS  247 

fresh  cells,  in  either  case  leading  to  the  production  of  free  and 
motile  endothelial  cells,  which  have  the  closest  resemblance  to 
the  large  hyaline  cells  of  the  blood,  though  they  may  be  much 
larger.  These  cells  are  most  active  and  important  phagocytes, 
especially  in  the  peritoneum.  They  have  also  the  power  of 
undergoing  organization,  especially  into  fibrous  tissue,  and  many, 
if  not  all,  of  the  fibroblasts  of  granulation  tissue  are  endothelial  in 
origin.  Further,  some  at  least  of  the  giant  cells  so  familiar  in 
chronic  inflammatory  processes  are  derived  from  the  endothelial 
cells  of  the  lymph  clefts  and  lymph  capillaries.  This  has  been 
proved  to  demonstration  by  Bergengriin  in  the  case  of  the  giant 
cells  in  leprosy.  Our  knowledge  of  the  origin  of  the  giant  cells  of 
tubercle  is  less  exact,  but  analogy  with  those  of  leprosy  would 
lead  us  to  infer  that  they  are  endothelial  also.  In  both  diseases 
the  giant  cells  are  most  important  phagocytes. 

Epithelial  cells  only  exceptionally  act  as  phagocytes.  We 
have  already  referred  to  the  ingestion  of  fat  by  the  columnar  cells 
of  the  intestine,  and  the  other  important  example  is  supplied  by 
the  epithelial  cells  lining  the  alveoli  of  the  lungs.  These  are 
flattened  plaques  under  normal  conditions,  but  in  the  presence  of 
an  irritant  they  become  cuboidal  or  columnar,  detach  themselves, 
or  bud  off  similar  cells,  and  are  powerful  phagocytes.  These  are 
the  dust  cells  so  frequently  seen  in  sputum. 

The  nature  of  the  cells  which  take  part  in  phagocytosis  is 
determined  to  some  extent  by  the  nature  of  the  irritant.  Thus, 
when  the  pyogenic  bacteria  are  ingested  it  is  usually  by  poly- 
nuclear  leucocytes,  whereas  it  is  extremely  rare  to  find  these 
cells  containing  tubercle  bacilli  in  the  tissues,  though  they  will 
take  them  up  readily  enough  under  the  artificial  conditions  of 
opsonic  experiments.  MetchnikofF  classifies  phagocytes  into  two 
groups — macrophages  and  microphages.  His  description  of  these 
cells  is  not  absolutely  clear,  but  in  general  the  microphages 
correspond  to  the  polynuclear  leucocytes,  and  the  macrophages  to 
the  large  hyaline  cells  of  the  blood  and  the  endothelial  cells  of 
the  serous  sacs  and  connective  tissues.  He  claims  that  the 
former  are  especially  concerned  with  the  phagocytosis  of  bacteria, 
the  latter  with  red  blood-corpuscles  and  similar  objects.  This 
distinction  is  not  a  valid  one,  since  endothelial  cells  are  ex- 
tremely active  phagocytes  for  bacteria,  and  polynuclear  cells  will 
ingest  red  corpuscles  with  great  readiness  when  provided  with 
a  suitable  opsonin  (see  Fig.  49).  It  would  appear  that  under 


248         PHAGOCYTES    OF   THE    PERITONEUM    AND    LUNG 

suitable  conditions  any  phagocyte  can  ingest  any  abnormal  body 
of  suitable  size. 

Under  certain  .conditions  there  may  be  traced  a  remarkable 
sequence  in  the  advent  of  various  phagocytes  to  an  infected  area, 
which  almost  suggests  a  symbiosis  of  the  nomadic  cells.  Thus, 
when  a  culture  of  a  bacteria  is  injected  into  the  peritoneum  of  the 
lower  animals,  a  very  definite  sequence  of  events  takes  place. 
The  peritoneal  fluid  normally  contains  some  small  mononuclear 
cells,  probably  of  endothelial  origin,  and  a  few  polynuclears. 
For  an  hour  or  so  after  the  injection  these  cells  are  diminished  in 
numbers,  and  the  eosinophiles  disappear  altogether.  The  exact 
cause  of  this  diminution  is  not  quite  clear.  It  may  be  due  to  the 
dilution  of  the  peritoneal  fluid  by  the  solution  injected,  or  to  the 
destruction  of  the  cells,  or  to  their  clumping  together  on  the 
omentum,  but  in  any  case  is  not  due  to  negative  chemotaxis.  For 
the  next  two  hours  or  so  there  is  a  gradual  increase  -of  the 
polynuclears,  at  the  end  of  that  time  an  influx  of  small  mono- 
nuclears,  until  in  about  six  hours  these  and  the  polynuclears  are 
present  in  approximately  equal  numbers.  After  this  the  mono- 
nuclears  (which  are  probably  budded  off  from  the  peritoneal 
cells,  and  are  thus  endothelial  in  origin)  gradually  become  larger, 
forming  what  Metchnikoff  calls  macrophages ;  then  the  fluid 
slowly  becomes  concentrated,  and  the  polynuclears  gradually 
disappear,  many  being  ingested  by  the  large  mononuclears. 
Finally  these  too  disappear,  but  it  takes  about  a  fortnight  for  the 
animal  to  revert  to  its  normal  condition.  Both  varieties  of  cells — 
endothelial  and  polynuclear — take  part  in  ingesting  the  cocci. 

Briscoe  has  shown  that  a  remarkable  series  of  processes  also 
occurs  when  organisms,  etc.,  are  injected  into  the  alveoli  of  the 
lungs.  It  varies  according  to  the  substance  injected,  and  we 
may  take  the  result  of  the  injection  of  the  potato  bacillus  as  an 
example.  In  the  first  hour  and  a  half  phagocytosis  is  very  active, 
the  bacilli  being  taken  up  exclusively  by  the  pre-existing  alveolar 
epithelial  cells.  Up  to  this  time  practically  no  polynuclears  have 
made  their  appearance,  but  they  now  commence  to  be  attracted 
into  the  alveoli,  where  they  occur  in  large  numbers  for  about 
twenty-four  hours.  They  take,  however,  but  a  small  share  in  the 
phagocytosis  of  the  bacteria,  and  are  themselves  taken  up  by  the 
alveolar  cells.  Some  proliferation  of  these  latter  cells  occurs, 
and  the  eosinophiles  increase  for  the  first  twenty-four  hours,  and 
then  gradually  diminish.  We  cannot  trace  the  reason  for  this 


PHAGOCYTOSIS  249 

sequence  of  phenomena,  but  it  is  evident  that  the  division  of 
labour  is  carried  to  a  high  pitch  amongst  the  phagocytes,  and 
that  there  must  be  some  controlling  influence  which  regulates  the 
appearance  of  the  cell  when  it  is  required. 

We  must  now  turn  to  a  discussion  of  the  importance  of  these 
facts  in  connection  with  immunity  as  it  appeared  to  the  patholo- 
gists  before  the  discovery  of  the  antibodies.  Much  of  this  is 


A     '^" 

l-    &  fi      > 


FIG.  50.  FIG.  51. 


FIG.  52. 
FIGS.  50  TO  52. — FROM  SCRAPINGS  FROM  THE  LUNGS  HALF  AN  HOUR,  Two 

HOURS,    AND    TWENTY-FOUR    HOURS    AFTER    THE     INJECTION     OF     POTATO 

BACILLI  INTO  A  BRONCHUS.     (From  films  lent  by  Dr.  Briscoe.) 

The  bacilli,  which  occurred  in  large  numbers  in  the  alveolar  cells  half  an  hour 
after  injection,  are  not  shown. 

mainly  of  historic  importance,  but  it  is  of  extreme  interest,  and  it 
is  to  the  controversy  which  occurred  between  the  cellular  and 
cellulo-humoral  schools  that  we  owe  much  of  our  knowledge  of 
the  processes  of  inflammation  and  of  the  functions  of  the  leuco- 
cytes. This  controversy  was  carried  out  with  great  skill  on  both 
sides,  and  was  the  means  of  suggesting  numerous  experiments 
of  much  beauty  and  ingenuity.  To  begin  with,  Metchnikoft's 


25O  OBJECTIONS    TO    THE    THEORY 

position  was  simple  and  logical.  He  pointed  out  that  in  mild 
and  non-fatal  infections  phagocytosis  usually  occurred,  and  the 
bacteria  could  be  readily  seen  inside  the  leucocytes,  whereas  in 
fatal  ones  little  phagocytosis  took  place,  if  any.  He  therefore 
enunciated  the  paramount  importance  of  the  process  in  immunity, 
and  at  one  time  considered  it  would  cover  the  whole  field  of  the 
phenomena. 

But  his  conclusions  did  not  pass  unchallenged,  and  the  sup- 
porters of  the  humoral  school  adduced  numerous  examples  of 
recovery  from  infection  where  little  phagocytosis  could  be 
observed,  and  went  farther,  and  showed  that  recovery  might 
occur  under  conditions  in  which  phagocytosis  was  impossible. 
The  best  experiments  of  this  sort  were  those  of  Baumgarten, 
which  were  repeated  by  Sanarelli.  These  observers  placed  non- 
virulent  bacteria  in  the  peritoneal  cavities  of  animals  enclosed  in 
bags  of  collodion  or  other  substances  which  would  permit  the 
free  diffusion  of  the  peritoneal  fluids,  but  would  prevent  the  access 
of  the  leucocytes,  and  they  found  under  such  conditions  that  the 
bacteria  were  completely  destroyed.  This  was,  of  course,  an 
example  of  bacteriolysis  of  a  type  with  which  we  are  now 
familiar.  Other  observers,  including  Metchnikoff  himself,  failed 
to  get  these  results ;  but  in  an  experiment  of  this  sort  a  positive 
result  is  of  more  value  than  a  negative  one.  It  is  possible,  for 
example,  that  the  walls  of  the  bags  which  Metchnikoff  prepared 
may  have  been  sufficiently  impermeable  to  prevent  the  access  of 
the  bacteriolytic  substances.  Then  other  observers  found  that 
bacteria  often  underwent  changes  indicative  of  death  and 
destruction  before  they  were  taken  up  by  the  phagocytes.  Thus 
Nuttall  found  that  when  attenuated  anthrax  bacilli  were  placed 
in  a  fine  tube  in  the  tissues  of  a  rabbit's  ear,  the  organisms  showed 
degeneration  forms  before  they  were  taken  up  by  the  leucocytes, 
and  thought  that  they  were  injured  by  the  serum  before  being 
ingested.  We  have  already  alluded  to  this  experiment  as  one  of 
the  starting-points  of  the  researches  on  the  alexins.  As  a  result 
of  experiments  such  as  this,  the  humoralists  relegated  phagocytosis 
to  a  part  of  quite  secondary  importance.  They  held  that  the 
injury  or  death  of  the  bacteria  by  the  humours  of  the  body  was 
the  important  factor,  and  admitted  only  that  the  phagocytes  acted 
as  scavengers  to  remove  the  dead  or  disabled  organisms.  To 
this  Metchnikoff  responded  by  allowing  a  leucocyte  to  take  up  a 
living  and  virulent  anthrax  spore,  and  then  isolating  the  leucocyte 


PHAGOCYTOSIS 


251 


and  planting  it  on  a  suitable  culture  medium,  on  which  the  cell 
died  ;  but  the  spore  survived,  showing  that  it  was  taken  up  with- 
out any  previous  injury.  He  also  traced  in  a  very  clear  and  full 
manner  the  steps  by  which  a  tubercle  bacillus  of  absolutely  normal 
appearance,  and  apparently  vigorous  and  healthy,  undergoes 


.d 


F1G.  53.— PROCESS  OF  ABSORPTION  OF  TUBERCLE  BACILLI  IN  GIANT  CELLS. 
a,  Unaltered  bacilli ;  b,  c,  d,  and  0,  various  stages  in  the  process. 

degeneration,  death,  and  absorption  in  the  giant  cell.  His  case 
was  proved  to  the  hilt  in  the  case  of  certain  bacteria,  whereas  his 
opponents  proved  theirs  in  others.  They  were  dealing  with 
immunity  of  different  types,  and  the  time  was  not  ripe  for  the 
solution  of  the  problem. 

The  views  of  another  school  which  sprang  up  at  this  point,  and 
which  attempted  to  reconcile  these  two  views,  are  of  more  impor- 


252  CELLULO-HUMORAL    THEORY 

tance,  in  that  they  approach  more  closely  to  the  modern  theory  of 
opsonic  immunity,  and,  indeed,  are  as  close  an  approximation  to 
it  as  could  have  been  formed  in  the  then  state  of  knowledge. 
They  were  as  follows  :  The  importance  of  phagocytosis  was 
recognized,  and  it  was  also  admitted  that  bacteria  were  frequently 
prepared  for  ingestion  by  dissolved  substances,  but  it  was  thought 
that  these  substances  emanated  from  the  leucocytes.  The  phago- 
cytes were  thought  to  produce  an  alexin  which  injured  the 
bacteria,  and  then  to  devour  them.  Baumgarten's  collodion-bag 
experiments  were  explained  by  supposing  that  the  leucocytes 
which  collected  round  the  bags  in  the  peritoneum  gave  off  alexin, 
which  diffused  through  and  was  sufficient  to  kill  the  leucocytes, 
though  more  slowly  and  with  more  difficulty  than  if  the  phago- 
cytes had  been  able  to  give  the  coup  de  grace.  In  dealing  with 
organisms  of  very  low  virulence  it  was  admitted  that  phagocytosis 
might  be  all-sufficient. 

Some  of  the  experiments  pointing  in  this  direction  may  be 
briefly  referred  to,  though  many  have  been  alluded  to  before  in 
the  chapter  on  the  complements.  Nuttall  continued  his  experi- 
ments on  the  destruction  of  anthrax  bacilli  by  a  comparison  of 
the  action  of  blood  and  serum,  and  found  that  the  latter  was 
enormously  the  more  powerful ;  and  this  he  explained  by  the 
assumption  that  the  protective  substances  are  given  off  in  the 
solution  of  the  leucocytes  which  occurs  in  the  process  of  clotting, 
and  many  other  experiments  were  forthcoming  in  support  of  this 
view.  But  the  most  beautiful  researches  were  those  of  Kanthack 
and  Hardy,  alluded  to  previously,  but  now  to  be  described  at 
greater  length.  When  anthrax  bacilli  are  placed  in  frog's  lymph 
and  examined  microscopically,  the  first  phenomenon  which  occurs 
is  the  approach  of  the  eosinophile  leucocytes  to  the  bacilli.  These 
cells  lose  their  granules,  and  at  the  same  time  the  bacilli  begin  to 
show  signs  of  degeneration,  the  inference  being  that  the  granules 
are  dissolved,  and  that  the  solution  acts  injuriously  on  the 
bacteria — i.e.,  is  alexin.  The  next  step  is  for  the  hyaline  cells 
to  approach  the  area  of  conflict,  and  to  fuse  with  the  eosinophiles 
to  form  a  plasmodium  around  the  bacilli.  Then  the  oxyphile 
cells  separate  themselves  from  the  plasmodium  and  move  away, 
and  then  the  hyaline  cells  can  be  seen  to  have  taken  up  the 
bacilli,  fragments  of  which  can  still  be  seen  within  them.  Lastly, 
a  number  of  cells  with  basophile  granulations  are  attracted,  but 
their  function  is  unknown.  It  is  obvious  that  there  is  here  a 


PHAGOCYTOSIS  253 

division  of  labour,  the  hyaline  cells  being  the  phagocytes  and  the 
eosinophiles  the  mother  cells  of  the  defensive  substances.  The 
granules  may  be  regarded  as  a  "  pro-enzyme  "  stage  of  alexin. 

It  must  not  be  thought  from  this  experiment  that  it  was  held 
that  the  eosinophile  cells  are  always  the  cells  which  secrete  the 


V  X    X    X    \ 

\         \         \         \         i 


"ft 


S      W      '  ft 


FIG.  54.— KANTHACK  AND  HARDY'S  EXPERIMENT.     (Original.) 

1-6,  An  oxyphile  leucocyte  attacking  a  thread  of  anthrax  bacilli ;  the 
figures  were  drawn  at  intervals  of  one  and  a  half  to  two  minutes,  the 
whole  sequence  occupying  twelve  minutes.  7-14,  A  thread  repeatedly 
attacked  by  three  oxyphile  leucocytes,  one  of  which  formed  a  plasmodium 
with  a  hyaline  cell  when  observations  were  commenced  ;  drawn  at 
intervals  during  a  period  of  one  hour.  15,  A  thread  attacked  by  a  plas- 
modium, consisting  of  an  oxyphile  and  a  hyaline  cell,  the  former  having 
lost  its  granules.  The  alteration  in  the  bacilli,  which  was  quite  clear  in 
the  specimens,  is  not  shown. 

The  whole  drawn  from  one  preparation,  the  first  series  immediately  after  it 
was  put  up,  the  second  after  half  an  hour,  and  number  15  after  two  hours. 

alexin.  This  is  certainly  not  the  case  in  man,  where  these  cells 
play  a  very  small  part  in  inflammatory  reactions  of  ordinary  type. 
Here  we  must  assume  that,  if  a  similar  process  occurs  at  all,  the 


254  ACTION    OF    "  CYTASE        IN    PHAGOCYTOSIS 

injurious  substance  is  provided  by  the  polynuclears,  which  thus 
play  both  parts. 

Kanthack's  experiment  is  the  best  and  most  direct  evidence  of 
the  extracellular  injury  of  bacteria  by  substances  derived  from 
the  leucocytes  occurring  as  a  preparation  for  phagocytosis. 

Metchnikoff  resisted  these  views  for  a  time,  but  soon  had  to 
admit  that  phagocytosis  is  not  the  only  factor  in  immunity ;  and 
he  then  altered  his  theory  in  an  ingenious  way,  and  regarded  the 
extracellular  injury  or  solution  of  organisms  as  being  essentially 
the  same  process  as  that  by  which  they  are  digested  after  being 
taken  in  by  the  phagocytes.  He  considers  that  bacteria,  red 
corpuscles,  etc.,  after  being  taken  in,  are  digested  by  the  action 
of  a  proteolytic  enzyme  which  he  calls  "  cytase  " — a  term  which 
has  been  already  alluded  to  as  a  synonym  for  complement  or 
alexin.  Of  this  there  are  two  sorts :  macrocytase,  which  is 
formed  by  the  macrophages,  and  which  digests  corpuscles,  cells, 
etc. ;  and  microcytase,  formed  by  the  polynuclears,  and  powerful 
against  bacteria.  Ordinarily  these  enzymes  are  restricted  to  the 
cells  which  form  them,  and  where  ingested  bodies  are  contained 
in  vacuoles,  these  latter  contain  a  solution  of  the  suitable  cytase  ; 
but  when  solution  of  the  phagocytes  occurs  the  cytase  is  set  at 
liberty,  and  may  then  exert  its  action  on  cells  or  bacteria  which 
are  lying  free.  Metchnikoff  regards  this  as  a  process  of  much  less 
importance  than  phagocytosis,  and  points  out  that  the  solution 
which  it  brings  about  is  rarely  complete :  thus,  when  bird's 
corpuscles  are  ingested,  they  are  entirely  absorbed,  nuclei  and 
all ;  whereas  when  they  are  acted  on  by  a  haemolysin  (which 
Metchnikoff  regards  as  a  macrocytase),  the  nuclei  remain.  This 
is  certainly  true  as  regards  the  action  of  most  sera  on  bacteria, 
solution  being  rarely  complete,  and  it  is  only  in  the  highly  potent 
sera  obtained  by  prolonged  immunization  to  certain  bacteria  that 
complete  disappearance  of  the  bacteria  occurs  as  a  result  of  the 
action  of  serum  ;  yet  when  taken  up  by  the  leucocytes  they  are 
digested  altogether,  sometimes  with  great  rapidity. 

The  difference  between  these  views  and  those  of  the  cellulo- 
humoralists  is  roughly  this  :  Metchnikoff  looks  upon  the  protective 
substance  as  a  digestive  enzyme  which  has  for  its  object  the 
transformation  of  the  foreign  cells,  etc.,  into  proteids  suitable  for 
the  nourishment  o£  the  phagocyte ;  whereas  most  bacteriologists 
regard  them  as  being  allied  to  the  toxins  rather  than  to  the 
enzymes,  and  as  being  specially  intended  for  the  defence  of  the 


PHAGOCYTOSIS 


255 


body  against  invaders.  The  point  is  one  of  theoretical  interest 
rather  than  of  practical  importance,  and  we  have  already  pointed 
out  that  the  complement  is  apparently  used  up  in  its  activity,  and  not 
set  free  to  attack  other  molecules,  as  is  the  case  with  the  enzymes. 
Another  minor  point  is  that  Metchnikoff  seems  to  regard  the 
setting  free  of  cytase  as  only  occurring  when  the  mother  cell  is 
dissolved,  whereas  most  of  the  bacteriologists  who  admit  the 
origin  of  alexin  from  leucocytes  regard  it  as  a  product  of  its 
secretory  activity.  The  point  has  been  referred  to  before. 
Metchnikoff  explains  the  phenomena  which  occur  in  immunized 


or. 


C 


FIG.  55. — PROCESS  OF  ABSORPTION  OF  ANTHRAX  BACILLI  IN  THE  LEUCO- 
CYTES OF  THE  PIGEON.     (Metchnikoff.) 

(Showing  various  stages  of  alteration  of  the  bacillus  whilst  in  the  protoplasm 
of  the  leucocytes.) 

as  opposed  to  normal  animals  in  this  way  :  We  will  take  the 
absorption  of  bird's  corpuscles  from  the  peritoneum  as  an 
example.  When  the  injection  takes  place  into  normal  animals, 
there  is  no  extracellular  destruction  of  the  corpuscles  (haemolysis), 
because  there  is  no  cytase  free  in  the  peritoneal  fluid,  no  cor- 
puscles having  been  broken  down  ;  the  corpuscles  are  taken  up 
by  the  phagocytes,  but  with  some  difficulty,  since  they  have  not 
been  prepared  in  any  way  for  the  process.  When  a  second  or 
third  injection  is  given,  some  haemolysis  occurs,  and  this  is 
because  the  cells  of  the  peritoneum  are  broken  down  by  the 
brusque  introduction  of  the  corpuscles ;  this  breaking  down  is 
termed  phagolysis,  and  is  regarded  as  being  a  necessary  pre- 
liminary to  the  liberation  of  the  cytase.  The  fresh  leucocytes 
which  arrive  now  proceed  to  ingest  the  corpuscles  with  great 


256  ANALOGY   WITH    DIGESTION 

avidity,  since  they  are  already  partially  digested.  We  should 
explain  the  phenomena  very  differently  :  the  haemolysis  is  a  result 
of  the  action  of  amboceptor  and  complement,  and  the  phagocytosis 
of  the  action  of  an  opsonin. 

The  explanation  of  bacteriolysis  and  haemolysis  by  means  of 
complement  and  amboceptor  might  appear  to  be  difficult  on  the 
theory  of  the  reference  of  the  whole  process  to  the  digestive 
action  of  the  phagocytes,  but  Metchnikoff  has  applied  the 
researches  of  Pawlow  in  a  very  ingenious  way  to  show  a 
parallelism  between  cytolysis  and  digestion.  It  will  be  remem- 
bered that  pancreatic  digestion  depends  upon  the  action  of  two 
substances  — an  enzyme,  protease,  which  occurs  in  the  pancreatic 
juice,  and  another  substance,  enterokinase,  which  occurs  in  the 
succus  entericus.  Metchnikoff  regards  the  protease  as  analoerous 

0>wJrV--tX>ft&tr^ 

with  cytase,  and  the  enterokinase  as  analogous  with  compleitieftt 
or  substance  sensibilatrice.  Delezenne  showed  (though  I  believe 
his  results  are  not  universally  accepted  by  physiologists)  that 
protease  has  no  power  of  attaching  itself  to  proteids,  whereas 
enterokinase  has  such  a  power,  and  the  substance  thus  sensitized 
can  then  be  attacked  by  protease.  This,  if  true,  is  exactly 
similar  to  the  action  of  amboceptor  and  complement.  We  may 
suppose,  then,  that  amboceptor  represents  some  substance  used 
by  the  phagocyte  to  assist  the  action  of  cytase  or  alexin  on  the 
bacteria,  etc.,  and  normally  retained  in  the  protoplasm. 

When  an  organism  which  is  easy  to  deal  with  is  injected  it  is 
taken  up  by  the  phagocytes  and  dealt  with  in  their  protoplasm, 
no  preparatory  action  being  necessary.  Under  other  circum- 
stances, when  the  infection  is  a  more  virulent  one,  some  of  the 
phagocytes  are  killed  and  dissolved,  and  their  digestive  enzymes 
escape,  and  partially  digest  the  bacteria,  which  are  then  ready  for 
phagocytosis.  When  there  is  a  balanced  contest  of  long  dura- 
tion another  substance  is  formed,  which,  under  normal  circum- 
stances, is  not  necessary  for  intracellular  digestion,  but  which 
facilitates  it  in  difficult  cases ;  this  also  may  escape  into  the 
juices,  and  still  further  facilitate  the  preparatory  stages  of  diges- 
tion. Lastly,  as  a  rarity,  enough  of  these  soluble  substances  may 
be  set  free  to  dissolve  the  bacteria  altogether,  and  render  phago- 
cytosis unnecessary.  To  Metchnikoff  cellular  digestion  and . 
nutrition  are  the  important  factors  in  immunity ;  extracellular 
action  is  a  less  important  and  occasional  phenomenon,  and  occurs 
mainly  or  entirely  as  a  preparation  for  phagocytosis.  His  theory 


PHAGOCYTOSIS  257 

is  logical,  complete,  and  well  supported  by  evidence,  but  it  does 
not  take  into  account  the  more  recent  work  of  Sir  Almroth 
Wright  and  his  followers,  and  this  now  calls  for  discussion  before 
the  role  of  phagocytosis  in  immunity  can  be  profitably  discussed 
further. 

It  may  be  admitted  that  Wright  did  not  discover  the  fact  that 
serum  may  aid  phagocytosis  by  acting  on  the  bacteria  ;  this  had 
been  already  shown  by  Denys  and  Leclef  in  1895,  by  Mennes 
and  by  Markl.  And  Neufeld  and  Rimpau  had  carefully  investi- 
gated the  same  property  in  the  serum  of  animals  immunized  to 
streptococci  and  pneumococci,  and  had  described  their  bacterio- 
tropic  substances,  which  are  apparently  identical  with  what  we 
now  know  as  thermostable  opsonins.  This  does  not  detract  in 
the  least  from  the  credit  due  to  Wright,  who  by  devising  a  simple 
quantitative  method  of  examination,  readily  applicable  in  clinical 
medicine,  made  a  very  great  advance  in  our  knowledge  of  the 
theory  of  the  subject,  and  has  added  a  most  important  and  useful 
method  of  examination  of  the  blood.  The  credit  for  the  intro- 
duction of  the  use  of  vaccines  in  the  treatment  of  established 
disease  (as  opposed  to  its  prevention)  is,  of  course,  due  to  him 
alone. 

The  name  opsonin  (opsono  =  I  cater  for,  I  prepare  for  food)  is 
given  to  substances  which  occur  in  the  serum  and  have  the  power 
of  preparing  bacteria  and  other  cells  for  ingestion  by  the  leuco- 
cytes, and  which  are,  or  are  held  to  be — for  there  is  no  absolute 
proof — different  from  the  substances  which  we  have  previously 
considered.  We  shall  discuss  this  question  of  identity  or  non- 
identity  subsequently,  and  shall  be  content  at  present  with  saying 
that,  whereas  bacteria  that  have  been  exposed  to  the  action  of 
alexin  are,  or  may  be,  obviously  injured,  a  bacterium  may  be 
saturated  with  opsonin  without  being  injured  in  the  least,  and 
may  still  retain  its  viability  and  virulence  uninjured. 

The  fundamental  experiments  of  Wright  and  Douglas  were  of 
this  nature,  and  they  are  easy  to  repeat  and  unimpugnable  in 
accuracy.  An  emulsion  of  leucocytes,  free  from  serum,  is  pre- 
pared by  receiving  blood  in  normal  saline  solution  containing 
citrate  of  soda,  centrifugalizing,  removing  the  supernatant  fluid 
and  replacing  it  with  saline  solution,  mixing  and  recentrifugalizing. 
This  process  must  be  repeated  until  all  trace  of  serum  is  removed, 
and  the  top  layer  of  the  deposit  is  then  pipetted  off,  and  will  be 
found  to  be  rich  in  leucocytes. 

17 


258  FUNDAMENTAL    EXPERIMENTS    ON    OPSONINS 

The  first  experiment  is  to  determine  whether  leucocytes  thus 
free  from  serum  are  able  to  ingest  bacteria.  To  this  end  they 
are  mixed  with  an  emulsion  of  staphylococci  or  tubercle  bacilli, 
enclosed  in  a  capillary  tube  and  incubated  at  37°  C.  for  a  quarter 
of  an  hour.  At  the  end  of  that  time  the  emulsion  is  expelled,  and 
films  are  prepared  and  stained  in  the  ordinary  way.  It  will  be 
found  that  the  leucocytes  have  taken  up  very  few  bacteria,  if  any. 
It  is  obvious,  therefore,  that  phagocytosis  goes  on  to  a  very  small 
extent  in  the  absence  of  serum.  Some  species  of  non-pathogenic 


£ 


"'\ 

'      «» 

" 


FIG.  56. — ON  THE  LEFT,  A  PORTION  OF  AN  OPSONIN  FILM  (OF  PNEUMOCOCCI)  ; 
ON  THE  RIGHT,  A  PORTION  OF  A  SIMILAR  FILM,  TAKEN  FROM  A  PREPARA- 
TION IN  WHICH  NO  SERUM  WAS  USED.  (Original.) 

bacteria  are  taken  up  well  in  the  absence  of  serum,  and  one  micro- 
coccus  which  I  have  met  with  was  not  ingested  under  any  circum- 
stances whatever. 

Secondly,  a  mixture  similar  to  the  above  is  prepared,  but  with 
the  addition  of  one  volume  of  serum,  so  that  the  mixture  consists 
of  an  equal  volume  each  of  leucocytic  emulsion,  bacterial  emul- 
sion, and  serum.  This  is  incubated  and  examined  as  above,  and 
it  will  be  found  that  many  bacteria  are  taken  up ;  the  number 
depends  on  the  thickness  of  the  emulsion  and  on  the  source  of  the 
serum,  but  if  the  former  be  rich  and  the  latter  potent  there  may 
be  an  average  of  twenty  or  even  far  more  per  polynuclear.  The 
bacteria  which  are  not  ingested  show  no  signs  of  digestion,  there 
being  no  loss  of  sharpness  of  contour  or  of  staining  activity. 


PHAGOCYTOSIS  259 

It  is  clear,  therefore,  that  serum  has  a  great  power  in  aiding 
phagocytosis.  Is  this  due  to  an  action  on  the  bacteria  or  to  a 
stimulation  of  the  leucocytes  ?  Two  experiments  show  that  the 
former  occurs ;  there  is  but  little  direct  evidence  for  or  against 
the  latter. 

In  one  experiment  Wright  (having  shown  that  the  power  of  the 
serum  is  destroyed  by  heating  it  to  55°  C.  for  thirty  to  sixty 
minutes,  or  to  60°  to  65°  C.  for  fifteen  minutes)  allowed  serum  to  act 
on  bacteria,  and  then  heated  the  mixture  until  the  activity  of  the 
serum  was  removed.  He  found  bacteria  thus  treated  were  taken 
up  readily.  This  must  have  been  due  to  an  action  which  the 
serum  had  exerted  on  them  before  it  was  heated,  and  any  action  on 
the  leucocytes  is  out  of  question,  since  they  were  only  acted  on  by 
heated  and  inactive  serum. 

Another  and  even  better  proof  of  the  same  fact  may  be  obtained 
by  acting  on  bacteria  with  serum,  centrifugalizing  and  removing 
all  trace  of  the  latter  by  repeated  washings  with  saline  solution. 
Bacteria  thus  treated  are  taken  up  with  great  readiness,  and  here 
no  free  serum  at  all  comes  into  contact  with  the  leucocytes.1 
Opsonin,  therefore,  combines  with  bacteria,  and  Bulloch  showed 
that  this  process  goes  on  at  ordinary  temperatures  and  at  o°  C.2 
Bacteria  which  have  once  been  acted  on  by  opsonin  ("opsonized") 
may  be  heated  to  60°  C.  for  five  hours,  and  are  still  assimilable  by 
leucocytes  ;  this  shows  that  they  are  profoundly  affected,  but  they 
may  be  absolutely  unchanged  in  appearance. 

There  is  no  method  by  which  an  absolute  measurement  of  the 
amount  of  opsonin  present  in  a  specimen  of  blood  can  be  made, 
but  comparative  measurements  can  be  made  easily  enough  by  the 
process  elaborated  by  Sir  Almroth  Wright.  In  order  to  do  this 
it  is  necessary  to  have  as  a  standard  either  the  serum  of  a  normal 
person,  or  preferably  a  mixture  of  sera  from  several  normal 
persons,  so  that  slight  individual  variations  or  abnormalities  may 
be  ruled  out.  The  emulsion  of  leucocytes  ("  cream  ")  is  prepared 
as  described  above,  and  the  emulsion  of  bacteria  made  by  stirring 
a  little  of  a  young  culture  of  the  organism  in  question  in  some 
saline  solution,  taking  care  to  remove  clumps  by  sedimentation  or 
centrifugalization.  When  tubercle  bacilli  are  being  used  it  is 
most  convenient  to  employ  dead  and  dried  bacilli,  which  are 

1  This  experiment  was  performed  by  Markl  in  1903,  using  plague  bacilli. 
-  This   is  not   altogether  confirmed  by    Ledingham's   more  recent  work, 
which  is  discussed  subsequently. 

I7—2 


260 


OPSONIC   TECHNIQUE 


ground  up  in  a  mortar  with  saline  solution  before  use.  The 
mixtures  of  leucocytes,  bacteria,  and  serum  are  made  in  capillary 
pipettes  mounted  with  an  indiarubber  nipple,  and  furnished  with 
a  unit  mark  about  i  inch  from  the  free  tip,  which  is  drawn  to 
a  fine  point.  The  process  is  as  follows  :  As  much  cream  as  will 
reach  to  the  unit  mark  is  drawn  into  the  pipette,  then  a  little  air 
(to  serve  as  an  index),  then  a  unit  of  the  emulsion,  another  bubble 
of  air,  and  finally  a  unit  of  serum.  These  are  then  blown  out  on 
to  a  glass  surface,  mixed  intimately  together,  sucked  into  the 
tube,  the  end  of  which  is  now  sealed.  The  tube  is  now  placed  in 
the  incubator  and  the  time  accurately  noted.  Then  the  process 


FIG.  57. — WRIGHT'S  CAPILLARY  PIPETTES,  AS  USED  IN  DETERMINATIONS 
OF  THE  OPSONIC  INDEX.  (Emery's  "  Clinical  Bacteriology  and 
Hsematology.") 

The  small  figure  shows  the  tip  magnified.  The  middle  figure  shows  the 
pipette  charged  with  leucocytic  cream  (in  this  case  two  volumes  are 
shown),  emulsion  of  bacteria,  and  serum.  In  the  lowermost  figure  these 
are  mixed  together  and  the  tip  sealed. 

is  repeated  in  exactly  the  same  way,  but  the  control  serum  is  used 
instead  of  that  which  is  being  investigated.  Each  pipette  is 
incubated  for  exactly  the  same  length  of  time,  rerribved  from  the 
incubator,  the  tip  broken  off,  the  contents  expelled,  and  films 
made.  These  are  obtained  in  a  suitable  way,  examined  under  the 
microscope,  and  the  number  of  bacteria  which  have  been  taken 
up  by  50  or  100  poly  nuclear  leucocytes  is  counted  in  each. 
Thus  we  may  find  that  in  the  control  specimen  (in  which 
healthy  blood  was  used)  there  are  300  bacteria  in  100  leucocytes ; 
in  this  case  we  say  the  "  phagocytic  index  "  is  3.  In  the  other 
specimen  (in  which  the  patient's  blood  was  used)  we  might  find 
150  bacteria  in  100  leucocytes,  giving  a  phagocytic  index  of  1*5. 


PHAGOCYTOSIS  26l 

We  see  in  this  case  that  the  patient's  blood  has  but  half  the 
opsonic  power  of  normal  blood ;  this  we  express  by  saying  that 
the  opsonic  index  is  0-5.  The  opsonic  index  is  obtained  by  dividing 
the  number  of  bacteria  found  in  a  certain  number  of  leucocytes 
in  the  films  made  with  the  patient's  serum  by  the  number  of 
bacteria  in  the  same  number  of  leucocytes  in  the  films  with  the 
control  serum,  and  expresses  the  phagocytic  power  of  the  patient's 
serum  as  compared  with  that  of  a  healthy  person.  It  is  not 
necessarily  an  exact  measure  of  the  amount  of  opsonin,  since  on 
dilution  of  a  serum  the  opsonic  index  falls  at  first  slowly  and  then 
more  quickly,  forming  a  flat-topped  curve  when  plotted  out  in  the 
usual  way  (see  Fig.  59,  p.  265). 

Other  methods  for  the  estimation  of  the  opsonic  index  have 
been  suggested,  and  require  some  mention.  In  the  earliest 
method — that  of  Leishman — the  patient's  blood  was  mixed 
directly  with  an  emulsion  of  the  bacteria  in  normal  saline  solution 
in  equal  parts,  and  a  drop  of  the  admixture  placed  on  a  slide, 
covered  with  a  cover-glass,  and  incubated  for  a  definite  time.  A 
control  specimen  was  prepared  in  a  similar  way,  using  normal 
blood.  After  the  incubation,  films  were  prepared  by  sliding  the 
cover-glass  off  the  slide,  stained,  and  a  count  made  as  in  the 
method  now  in  use.  A  similar  but  rather  better  method  is  also 
employed,  and  is  extremely  convenient  in  some  cases.  The 
bacterial  emulsion  is  prepared  as  above,  the  organisms  being 
suspended  in  normal  saline  solution  containing  sodium  citrate. 
A  mixture  of  this  emulsion  and  of  the  patient's  blood  in  definite 
amounts  (usually  equal  parts)  is  prepared,  sucked  into  the  pipette 
(the  tip  of  which  is  sealed),  and  incubated  for  a  quarter  of  an 
hour  or  twenty  minutes.  The  process  is  the  same  as  Leishman's 
except  that  the  mixture  is  incubated  in  a  pipette,  and  not  between 
slide  and  cover-glass.  A  control  is  also  prepared,  using  the  same 
emulsion  and*  normal  blood,  and  is  also  incubated  for  a  quarter 
of  an  hour.  At  the  end  of  this  period  films  are  prepared,  and 
the  process  finished  in  the  ordinary  way. 

This  method  is  theoretically  more  accurate  as  a  test  of  the 
phagocytic  activity  of  the  patient's  blood  as  compared  with  normal 
blood  than  is  the  opsonic  index  as  determined  in  the  ordinary 
way,  in  which  leucocytes  from  the  same  source  are  used  in  both 
determinations — i.e.,  in  that  of  the  patient's  serum  and  in  that 
of  the  control.  Thus,  if  in  any  case  the  leucocytes  were  so 
injured  that  they  had  very  little  phagocytic  power,  the  opsonic 


262  OPSONIC   TECHNIQUE 

index  as  determined  by  Wright's  method  might  nevertheless  be 
normal ;  yet  this  blood  might  have  but  little  power  of  destroying 
bacteria  which  gain  access  thereto.  There  is  some  experimental 
evidence  that  alterations  in  the  power  of  the  leucocytes  do 
actually  occur  ;  thus  Shattock  and  Dudgeon,  in  some  experiments 
with  granules  of  melanin  (which,  like  bacteria,  require  to  be 
opsonized  before  they  can  be  taken  up  by  leucocytes),  found  that 
either  more  or  less  might  be  taken  up  by  the  patient's  leucocytes 
as  compared  with  normal  ones,  using  the  same  serum  in  all  cases. 
The  numbers  varied  between  0-46  and  2*9,  taking  the  normal 
number  as  unity.  It  must  be  pointed  out  that  this  method  does 
not  give  the  opsonic  index  of  the  serum,  and  that  in  cases,  e.g., 
in  which  a  low  result  is  obtained  it  affords  no  information  as  to 
whether  the  leucocytes  or  the  serum  is  at  fault,  or  both.  Further, 
there  is  a  possible  error  owing  to  the  possible  difference  in  the 
number  of  the  leucocytes  in  the  unit  volume  of  the  two  specimens 
of  blood.  Where  the  patient  has  a  leucocytosis — and  this  is  very 
common  in  the  type  of  case  in  which  opsonic  estimations  are 
required — the  difference  may  be  very  great.  The  result  of  this 
has  not  been  fully  elucidated,  but  it  is  obvious  that  where  the 
bacterial  emulsion  is  not  very  thick  the  number  available  per 
leucocyte  is  very  different  in  the  two  cases.  This  is  a  point 
worthy  of  consideration  in  the  determination  of  the  opsonic  index 
by  Wright's  method.1  When  the  bacterial  emulsion  is  very 
dilute  a  large  error  is  introduced,  and  even  if  very  large  numbers 
of  leucocytes  are  counted  the  results  are  untrustworthy.  The 
best  results  theoretically  would  be  obtained  where  the  emulsion 
was  so  thick  that  every  leucocyte  would  take  up  as  many  bacteria 
as  it  was  capable  of  doing  in  the  given  time.  This  is  impracticable, 
however,  as  the  labour  in  counting  leucocytes  containing  very 
many  bacteria  is  great,  and  the  error  in  counting  is  also  large. 
Probably  the  best  results  are  obtained  where  the  phagocytic 
index  in  the  control  is  about  4,  and  it  is  a  good  plan  to 
perform  an  orientating  experiment  to  determine  the  appropriate 
strength  of  the  emulsion  before  commencing  a  large  series  of 
opsonic  determinations. 

1  It  has  been  investigated  by  Ruth  Tunnicliffe,  who  finds  no  very  great 
differences  in  a  series  of  estimations  in  which  the  bacteria  (diphtheria  bacilli) 
varied  from  125,000  to  1,000,000  per  cubic  millimetre,  all  the  other  factors 
being  constant  ;  and  by  Walker,  who  finds  that  the  index  rises  greatly  if  a 
thicker  emulsion  is  employed. 


PHAGOCYTOSIS  263 

Another  modification  of  Wright's  method,  introduced  by 
Simon,  concerns  the  method  of  counting  only.  A  large  number 
of  leucocytes  are  counted,  and  are  classified  simply  into  those 
that  contain  bacteria  and  those  that  are  free.  Of  course,  the 
emulsion  must  not  be  too  thick,  or  practically  all  the  leucocytes 
will  have  taken  some  up.  The  process  is  repeated  with  the 
control,  and  the  results  compared;  thus,  if  in  the  control  film 
25  per  cent,  of  leucocytes  were  empty,  and  in  the  patient's  film 
50  per  cent.,  the  index  would  be  §£  =  0-5.  A  comparison  of  the 
results  obtained  by  this  method  and  by  careful  counting  show 
that  they  are  fairly  comparable,  and  the  process  may  be  used 
where  it  is  only  necessary  to  determine  whether  the  index  is 
high  or  low. 

Another  and  more  important  method  is  that  of  dilution  or 
extinction,  as  introduced  by  Dean  and  by  Klien.  It  is  especially 
useful  in  the  case  of  bacteria,  such  as  B.  typhosus  and  V.  cholera, 
which  are  dissolved  by  fresh  serum  when  but  slightly  diluted. 
Further,  when  an  attempt  is  made  to  determine  the  opsonic  index 
to  the  former,  and  the  pipette  is  incubated  for  but  five  minutes, 
numerous  shadows  and  partially  digested  bacilli  are  seen  within 
the  leucocytes,  thus  introducing  a  new  and  very  important  error. 
In  order  to  avoid  this,  Klien  determines  the  degree  of  dilution  of 
the  serum  necessary  for  the  complete  extinction  of  its  opsonic 
action.  In  preparations  in  which  no  serum  is  used  the  phagocytic 
index  is  usually  below  0*5,  and  the  serum  to  be  tested  is  diluted 
until  the  degree  of  dilution  is  found,  which  gives  a  phagocytic 
index  no  higher  than  this.  Working  by  this  method,  Klien 
obtained  results  very  different  from  those  obtained  by  Wright's 
method.  In  the  process  of  immunization  of  a  rabbit  the  index 
(by  the  latter  method)  remained  low,  varying  only  between  0-82 
and  1-65,  whereas  by  the  process  of  dilution  it  was  seen  to  be 
actually  greatly  raised.  Before  the  commencement  of  the  im- 
munization the  opsonic  power  of  the  serum  was  extinguished 
when  the  latter  was  diluted  thirty  times,  whereas  afterwards  it 
did  not  disappear  until  diluted  3,072  times.  It  appears  clear  that 
in  the  case  of  bacteria  like  this  the  results  obtained  by  Wright's 
method  are  quite  misleading.  Klien  states  that  the  bacterial 
emulsion  should  be  a  thick  one,  and  should  be  of  about  the  same 
density  in  successive  experiments,  if  these  are  to  be  comparable. 
The  main  objection  to  this  method  is  its  tediousness:  many 
pipettes  have  to  be  prepared,  and  many  films  examined. 


264 


METHOD    OF    EXTINCTION 


It  has  an  advantage  over  Wright's  method  even  in  cases  in 
which  the  bacteria  are  not  dissolved  in  the  serum  or  leucocytes, 
in  that  it  provides  a  definite  measure  of  the  amount  of  opsonin 
present,  which  the  ordinary  method  does  not  do,  as  is  shown  by 
the  fact  that  the  opsonic  index  of  a  mixture  of  equal  parts  of 
serum  and  normal  saline  solution  is  more  than  half  that  of 
undiluted  serum. 

An  enormous  number  of  opsonic  determinations  have  been 
carried  out,  and  the  results  have  been  of  extreme  interest.  It  is 


3000 

2,800 

2,600 

2,400 

2.200 

2,000 

1.800 

1.600 

1,400 

1,200 

1.000 

800 

600 

400 

200 


IB   17    19  21   23  25  27  29  31    2    4    6    8    10    12    14 

*         =  Leucocytes  per  cubic  mm.  (figures  at  right  of  chart). 
—  —  —  •  —  —  -  =  Opsonic  power  of  serum. 

• =  Bacteriolytic  power  of  serum. 

t--  —  -   =  Agglutinative  power  of  serum. 

FIG.  58. — INFLUENCE  OF  INOCULATION  OF  TYPHOID  VACCINE  ON  THE  OPSONIC 
POWER  OF  THE  SERUM  OF  A  RABBIT,  AS  SHOWN  BY  THE  DILUTION 
METHOD.  (After  Klien.) 

found  that  the  indices  of  healthy  persons  is  approximately  the 
same,  and  does  not  vary  much  from  day  to  day.  In  the  case  of 
tubercle  a  very  large  number  of  determinations  of  the  indices  of 
normal  persons  have  been  made,  and  it  is  found  that,  with  one 
or  two  exceptions,  due  perhaps  to  accidental  errors  in  technique, 
they  all  lie  between  o\S  and  1-2,  taking  i  as  a  standard.  In  reality 
they  agree  very  much  more  closely  than  this,  for  the  great 


PHAGOCYTOSIS 


majority  lie  much  nearer  to  i.  When  the  estimations  are  carefully 
carried  out,  very  few  will  be  found  below  0*95  or  above  i'O5-  We 
may  regard  the  opsonic  index  for  a  given  organism  as  a  definite 
quantity  in  a  healthy  person.  Some  sera  are  lower  or  higher  than 
others,  but  the  difference  is  but  slight,  and  the  index  of  the  same 
person  is  found  to  show  but  slight  daily  variations  as  long  as  he 
remains  in  good  health.  A  few  observations  go  to  show  that  the 
index  is  slightly  lowered  in  persons  who,  without  being  ill,  are  in  a 


Serum      4: 1 


3:2         2:3 


C4      N.Safine 


FIG.  59. — SHOWING  EFFECT  OF  DILUTION  OF  NORMAL  SERUM  ON  THE 
NUMBER  OF  BACTERIA  TAKEN  UP.     (Original.) 

state  of  lowered  vitality,  and  that  the  onset  of  a  mild  disease, 
such  as  a  cold,  may  cause  a  fall  in  the  index  to  tubercle  or  other 
disease. 

When  the  patient  suffers  from  a  disease  due  to  a  given  organism, 
and  his  index  is  tested  against  this  organism,  the  results  obtained 
are  of  extreme  interest.  Taking  the  acute  diseases  first,  we  find 
that  as  a  rule  the  index  is  low  at  the  commencement  of  the  illness, 
and  that  it  rises,  either  gradually  or  suddenly,  when  recovery 
takes  place ;  and  in  some  cases  there  is  a  definite  correlation 
between  the  course  of  the  index  and  that  of  the  disease.  For 
example,  Macdonald  has  shown  that  in  an  attack  of  pneumonia 
the  opsonic  index  of  the  patient's  serum  to  the  pneumococcus 
remains  at  a  constant  low  level  until  the  crisis  is  reached,  when  it 
shows  a  sudden  rise,  attaining  a  point  above  the  normal  level.  It 
remains  elevated  for  a  short  time,  and  then  relapses  to  normal  or 
below  normal.  It  is  difficult  to  believe  that  the  rise  in  the  opsonic 
index  and  the  consequent  increase  in  phagocytosis  which  we 


266 


OPSONIC    INDEX    IN    ACUTE    INFECTIONS 


should  expect  to  be  caused  thereby  is  not  the  cause  of  the  crisis 
and  the  patient's  recovery.  The  short  duration  of  the  high  level 
of  the  index  is  interesting,  as  we  know  that  the  immunity  left 
after  an  attack  of  pneumonia  is  but  temporary. 

A  gradual  rise  of  the  index  often  takes  place  in  staphylococcic 
diseases — e.g.,  boils  ;  and  when  the  index  is  traced  from  day  to  day, 
it  may  be  seen  that  it  is  low  to  begin  with,  during  the  onset  and 
increase  of  lesion,  but  that  it  rises  more  or  less  gradually  until  it 

Days  of  disease 

I     2    5    4     6    6    7    8    9    10    II    12   13   14   15 


1-6  ^ 

1-6  /\ 

1-4  /     \ 

B  /  V  - 


FIG.  60. — TYPES  OF  REACTION  OF  THE  OPSONIC  INDEX  IN  PNEUMOCOCCIC 
INFECTION.     (After  Eyre.) 

(a)  Immediate,  as  seen  in  mild  diseases ;   (b)  delayed  ;  and   (c)  progressive 
decline,  as  seen  in  severe  and  fatal  infections. 

reaches  a  point  well  above  normal.  At  the  same  time  the  disease 
begins  to  improve,  suggesting  again  that  the  phagocytosis  de- 
pendent on  the  amount  of  opsonin  in  the  blood  is  the  actual 
cause  of  the  recovery.  Very  many  observations  of  this  type  have 
now  been  made  with  many  organisms,  and  as  a  general  rule  we 
may  say  that  in  acute  diseases  (excluding  tubercle)  the  index  is,  as 
a  rule,  low  during  the  onset  and  culmination  of  the  disease,  and 
raised  during  involution  and  recovery.  Exceptions  may  be  met 
with,  but  the  sequence  of  events  happens  too  often  to  be  a  mere 
coincidence  (see  Figs.  60,  61,  63,  and  64). 


PHAGOCYTOSIS 


267 


Hence  the  opsonic  school  of  immunity  has  formed  a  theory 
which  may  be  enunciated  as  follows :  The  immunity  to  certain 
organisms  (not  to  all)  depends  on  phagocytosis,  and  this  can  only 
take  place  in  virtue  of  the  preparation  of  the  organism  by  the 
action  of  opsonin.  Where  this  substance  is  present  in  normal 
amount  the  person  is  sufficiently  immune  to  resist  ordinary 
infections;  but  if  for  any  reason  the  amount  is  lowered  or  the 


2 

1-8 
1-8 
1-7 
1-6 
1-5 
14 
1-3 
1-2 
l-l 
1-0 
9 
8 
7 
6 
5 
4 
3 
2 


FIG.  61. — OPSONIC  INDEX  IN  DIPHTHERIA.     (Tunnicliffe.) 

infection  very  virulent,  phagocytosis  cannot  occur,  and  the  disease 
progresses.  There  is  then  a  new  formation  of  opsonin,  just  as 
there  is  of  other  antibodies,  and  this  goes  on  until  there  is 
sufficient  to  sensitize  all  the  bacteria  and  render  them  amenable  to 
phagocytosis,  when  recovery  occurs.  When  this  does  not  take 
place  the  patient's  phagocytes  cannot  ingest  the  bacteria,  and  the 
disease  progresses. 

There  is  one  assumption  that  will  require  critical  consideration 
subsequently,  and  that  is  that  opsonin  is  an  antibody. 

The  behaviour  of  the  index  in  chronic  infections  is  different, 


268  OPSONIC    INDEX    IN    CHRONIC    INFECTIONS 

and  is  difficult  to  explain  on  the  opsonic  theory  of  immunity.  In 
a  chronic  staphylococcic  lesion,  such  as  acne,  the  index  may  be 
low,  normal,  or  high,  and  this  is  also  the  case  to  a  most  marked 
extent  in  tuberculosis.  Wright  classified  the  cases  of  this  disease 
into  two  groups:  (i)  strictly  localized  tubercle,  such  as  lupus, 
mild  glandular  cases,  tuberculous  abscesses,  etc. ;  (2)  cases 
associated  with  constitutional  disturbances.  In  the  former  he 
found  the  index  uniformly  low  (from  0-13  to  o!88),  whereas  in 
the  latter  there  was  great  variation,  the  index  being  below  normal, 
or  as  high  as  2  or  more.  Further  researches,  however,  have 
not  confirmed  this,  and  the  indices  of  patients  with  lupus  will 
often  be  found  very  high.  As  a  rule,  however,  the  patients  with 
localized  tubercle,  if  kept  at  rest  in  bed,  will  be  found  to  have 
a  constant  index,  whereas  in  those  with  a  progressive  disease  it 
will  be  found  to  vary  from  day  to  day,  being  often  very  high. 

These  variations  are  attributed  to  auto-inoculation — i.e.,  to  the 
discharge  from  the  lesion  of  a  few  bacteria  or  of  a  small  dose 
of  bacterial  toxin,  which  makes  its  way  into  a  region  suitable 
for  the  elaboration  of  a  further  amount  of  opsonin,  acting  just 
as  an  injection  of  a  vaccine,  and  causing  a  negative,  followed  by 
a  positive  phase.  When  the  patient  is  kept  absolutely  at  rest  in 
bed  this  does  not  occur,  or  only  to  a  comparatively  slight  extent, 
and  the  index  is  more  or  less  steady.  If,  however,  the  patient  be 
allowed  to  exert  himself,  even  slightly,  or  if  the  lesions  are 
gently  massaged,  specific  substances  are  set  free,  auto-inoculation 
occurs,  and  the  index  exhibits  its  characteristic  oscillations.  It 
is  also  dependent  to  some  extent  on  the  temperature,  as  has  been 
shown  by  Inman  and  others,  tending  (in  phthisis)  to  fall  with  a 
rise  of  temperature,  and  vice  vevsz.  In  general,  a  fluctuating 
temperature  accompanies  a  subnormal  index,  a  rise  occurring 
when  the  oscillations  become  less.  The  injection  of  a  bacterial 
vaccine  may  cause  a  rise  of  temperature,  especially  if  the  amount 
is  large,  but  does  not  always,  and  should  not,  do  so. 

In  chronic  infections  a  high  opsonic  index  does  not  necessarily 
imply  that  a  patient  is  doing  well.  In  general  tuberculosis  the 
index  is  often  normal  or  elevated,  and  a  rise  may  occur  just 
before  death.  This  is  also  the  case  in  acute  infections,  such  as 
erysipelas,  in  which  a  sudden  and  great  elevation  may  immediately 
precede  the  fatal  issue  (Fig.  63). 

These  results  are  difficult  to  harmonize  with  the  opsonic  theory, 
but  Wright  points  out  that  it  is  not  sufficient  for  there  to  be 


PHAGOCYTOSIS 


269 


enough    opsonin    in    the    blood;    it    must    reach    the    diseased 
tissue. 

Some  observations  go  to  show  that  it  may  be  unable  to  do 
this  under  certain  conditions.  Thus  Bulloch  found  the  liquor 
puris  from  a  staphylococcic  abscess  entirely  devoid  of  opsonin  to 
staphylococci.  This  might  have  been  due  to  absorption  by  the 
bacteria  in  the  pus,  so  he  cleansed  the  abscess,  and,  taking  the 
first  few  drops  which  collected,  found  them  also  very  deficient 
in  opsonin.  It  appears,  therefore,  that  this  substance,  though 
present  in  the  blood,  was  unable  to  make  its  way  through  the 


DATE 

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FIG.  62. — SHOWING   INVERSE   RELATIONSHIP   BETWEEN  TEMPERATURE  AND 
OPSONIC  INDEX  IN  PHTHISIS.     (Inman.) 

The  continuous  line  shows  the  temperature. 

wall  of  the  abscess  to  the  place  where  it  was  wanted.  Again, 
Wright  has  shown  that  the  serous  fluid  in  cases  of  tuberculous 
pleurisy  and  peritonitis  is  very  low  in  opsonin  as  compared  with 
the  circulating  blood,  and  has  made  use  of  this  fact  as  a  means 
of  diagnosis.  It  must  be  obvious  that  in  the  case  of  an  extra- 
vascular  object  like  a  tubercle,  and  especially  a  caseous  mass, 
that  a  slight  alteration  in  the  opsonic  index  of  the  blood  can 
have  but  a  slight  immediate  effect ;  any  beneficial  effect  of  a 
high  index  must  be  slow  in  manifesting  itself.  To  remedy  this, 
Wright  attempts  to  flush  the  morbid  tissues  with  blood  or  lymph 


270 


SPECIFICITY    OF   OPSONINS 


by  diminishing  the  viscosity  of  the  blood  by  the  exhibition  of 
citrates  and  other  anticoagulants,  by  the  use  of  hot  applications, 
and  by  Bier's  method  of  congestion. 

The  first  point  which  arises  in  a  discussion  of  the  opsonic 
theory  deals  with  the  specificity  of  the  opsonins  themselves.  Are 
we  to  imagine  that  there  is  a  specific  opsonin  to  each  organism, 
and  that  during  the  process  of  immunization  this  increases, 
whilst  the  others  remain  constant  ?  Unless  this  is  the  case,  the 
theory  fails,  for  we  know  that  immunity  is  specific. 


101 
100 
99 


2-5 


1-5 


Day 
7   8 


$  10  II  12  13 


FIG.  63. — BEHAVIOUR  OF  THE  OPSONIC  INDEX  IN  A  MILD  (a)  AND  SEVERE 
(6)  CASE  OF  ERYSIPELAS.     (After  Tunnicliffe. ) 

The  latter  shows  the  preagonal  rise  ;    the  broken  line  in  the  first  chart 
indicates  the  temperature. 


The  question  may  be  investigated  in  two  ways — by  absorption 
of  the  opsonins  and  by  comparison  of  different  sera. 

The  first  method  was  employed  by  Bulloch  and  Western,  who 
added  an  emulsion  of  tubercle  bacilli  to  normal  serum,  and  found 
but  a  slight  reduction  of  the  opsonic  index  to  staphylococci, 
suggesting  the  difference  of  the  two  opsonins.  But  these 
results  have  not  been  confirmed  by  later  writers,  and  it  is  quite 
certain  that  a  sufficient  amount  of  tubercle  bacilli  will  remove 
practically  the  whole  of  the  opsonin  to  staphylococci.  These 
experiments  tend,  therefore,  to  show  that  the  opsonins  are  not 


PHAGOCYTOSIS  27! 

specific,  and  that  any  immunity  due  to  them  would  be  a  general 
one. 

The  second  method  is  by  a  comparison  of  various  sera  in  their 
action  on  various  organisms.  For  example,  we  may  take  two 
sera,  and  compare  them  in  their  action  on  tubercle  bacilli  and  on 
staphylococci.  If  we  find  uniformly  that  a  serum  which  is  low 
to  one  is  also  low  to  the  other,  it  will  tell  strongly  against  the 
theory  of  specificity.  This,  however,  is  what  we  do  not  find, 
and  it  is  quite  usual  to  discover  that  a  serum  which  is  very  low 
to  the  tubercle  bacillus  as  compared  with  a  normal  control  has 
a  normal  index  to  staphylococci  as  compared  with  the  same 
control.  Of  this  there  can  be  no  doubt.  Further,  after  the 
injection  of  a  vaccine  composed  of  the  dead  bodies  of  certain 
organisms,  it  is  usual  to  find  the  opsonic  index  to  that  organism 
rise,  whereas  to  others  it  remains  unaltered.  This  tends  very 
strongly  to  show  that  opsonins  are  specific  bodies. 

Quite  similar  results  are  seen  when  the  behaviour  of  the 
opsonic  index  to  two  or  more  bacteria  is  followed  from  day  to 
day  in  a  patient  suffering  from  an  infection  by  one  of  them. 
Thus,  in  a  patient  who  was  recovering  from  a  severe  furuncle 
the  index  to  staphylococci  and  tubercle  bacilli  was  observed,  with 
the  result  shown  in  Fig.  64. 

Here  we  may  regard  the  tubercle  opsonin  as  being  normal 
throughout,  the  slight  variations  met  with  being  well  within  the 
range  of  experimental  error.  The  index  to  staphylococci,  on  the 
other  hand,  ranged  between  0*4  and  1*35,  and  showed  a  general 
parallelism  with  the  amelioration  in  the  patient's  condition.  It  is 
obvious  that  the  two  indices  are  not  due  to  the  presence  of  a 
single  opsonin. 

Reverting  to  the  saturation  experiments,  we  may  perhaps 
explain  them  as  follows  :  Any  opsonin  can  prepare  any  bacterium 
for  phagocytosis  if  it  combines  with  it ;  but  there  are  different 
opsonins,  with  very  different  degrees  of  affinity  for  different 
bacteria.1  Thus  we  may  suppose  the  tubercle  opsonin  to  have  a 
powerful  affinity  for  the  tubercle  bacillus,  a  slight  one  for  the 
staphylococcus,  so  that  the  addition  of  a  few  tubercle  bacilli  will 

1  It  now  seems  fairly  clear  that  the  explanation  of  these  experiments  is  that 
fixation  of  complement  (which  in  this  case  acts  as  an  opsonin)  takes  place. 
Normal  serum  contains  an  amboceptor  (  =  thermostable  opsonin)  to  staphylo- 
cocci, though  in  small  amount ;  and  this,  when  combined  with  staphylococci, 
will  attract  all  the  opsonin  to  it,  the  staphylococcus  opsonin  most  powerfully. 


272 


SPECIFICITY   OF   OPSONINS 


remove  it  from  a  sample  of  serum,  whereas  a  large  number  of 
staphylococci  are  required.  In  this  case  opsonins  will  have  a 
sort  of  modified  specificity  comparable  with  that  of  the  agglutinins 
for  the  coli  group,  and  this  appears  to  harmonize  Bulloch's  results 
with  those  of  later  observers. 

An  example  of  this  selective  absorption  of  opsonins  may  be 
given,  chiefly  to  illustrate  the  methods  employed  in  this  class 


1-5 
14 
1-3 
1-2 

II 

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•6 
•5 
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FIG.  64. — BEHAVIOUR  OF  OPSONIC  INDEX  TO  STAPHYLOCOCCI  AND  TUBERCLE 
BACILLI  DURING  NATURAL  RECOVERY  FROM  AN  ATTACK  OF  FURUNCU- 
LOSIS.  (Original.) 

of  experiment.  A  specimen  of  normal  serum  was  mixed  with  an 
equal  amount  of  very  thick  emulsion  of  staphylococci,  kept  at 
37°  C.  for  one  hour,  and  then  centrifugalized  until  all  the  cocci  were 
removed,  leaving  the  fluid  (a).  A  second  amount  of  serum  was 
treated  similarly,  but  the  staphylococci  emulsion  was  diluted 
100  times  (b).  It  was  hoped  that  the  staphylococcic  opsonin 
would  be  completely  removed  from  the  first,  and  only  partially 
removed  from  the  second  specimen.  This  was  tested  as  follows  : 


PHAGOCYTOSIS  273 

Four  experiments  were  carried  out,  in  each  of  which  the  fluids 
(£  unit  of  each)  were  mixed  with  i  unit  of  leucocyte  "cream," 
and  of  a  fine  emulsion  of  staphylococci,  incubated,  and  films 
prepared  and  counted.  Thus  : 

Staphylococci  in     ,,     l 
50  Leucocytes. 

1.  Normal  serum  +  normal  saline          ...         ...  170  1*0 

2.  ,,  ,,      +  supernatant  fluid  (b)          ...  125  o'6g 
3-         ,,           ,,      -f-           ,,              ,,     (*)          ...               55  °'21 
4.  Normal  saline  -f  normal  saline          ...         ...               24 

These  fluids  were  then  tested  in  exactly  the  same  way  with 
regard  to  their  action  on  tubercle  bacilli.  Thus : 

Tubercle  Bacilli     ,    , 
in  50  Leucocytes, 

1.  Normal  serum  +  normal  saline          ...         ...  145  ro 

2.  ,,  ,,      -f  supernatant  fluid  (b)          ...  132  0*93 
3-         ,,                   +           ,,              ,,     (a)          ...              go  0-6 
4.  Normal  saline  +  normal  saline          ...         ...                 9 

Here  it  is  obvious  that  the  staphylococci  have  removed  the 
staphylococcic  opsonins  more  powerfully  than  the  tubercle 
opsonin.  The  strong  emulsion  removed  80  per  cent,  of  the 
former  and  only  40  per  cent,  of  the  latter. 

A  striking  example  of  the  fact  that  there  is  more  than  one 
sort  of  opsonin  is  supplied  by  observations  on  the  haemopsonins. 
Most  specimens  of  blood-serum  are  unable  to  act  as  opsonins 
for  the  red  corpuscles,  which  are  not  taken  up  by  the  leucocytes 
under  the  ordinary  conditions  of  opsonin  investigation  in  vitro  ; 
but  some  specimens  do  possess  the  power  of  opsonizing  red 
corpuscles.  It  is  obvious,  therefore,  that  haemopsonin  is  not  the 
same  as  bacteriopsonin. 

We  must  now  discuss  the  nature  of  these  opsonins.  Are  they 
familiar  substances  (e.g.,  complements  or  amboceptors)  mas- 
querading under  a  new  name,  or  are  they  essentially  different  ? 
And  if  so,  are  they  antibodies,  or  are  they  allied  to  other  protective 
substances,  such  as  the  alexins  of  the  cellulo-humoralists  or  the 
cytases  of  Metchnikoff  ?  This  is  an  extremely  difficult  subject, 
and  one  which  has  not  yet  been  satisfactorily  solved. 

The  main  evidence  in  favour  of  the  view  that  they  are  specific 

1  The  indices  given  are  corrected  by  the  deduction  of  the  number  of  bacteria 
taken  up  spontaneously  (Expt.  4)  from  each  of  the  totals.  This  may  be 
termed  the  corrected  opsonic  index,  and  ought  to  be  given  where  great 
accuracy  is  required. 

18 


274 


RESULT    OF    INJECTIONS   OF   VACCINES 


antibodies  is  derived  from  a  study  of  their  behaviour  when  a 
patient  is  inoculated  with  their  specific  antigens.  If,  for  instance, 
a  patient  with  a  low  index  for  tuberculosis  is  inoculated  with  a 
small  dose  of  new  tuberculin  (say  y^^  milligramme),  consisting 
of  the  dead  bodies  of  the  tubercle  bacilli,  a  very  definite  train  of 
phenomena,  closely  comparable  to  the  results  of  an  injection  of  diph- 
theria toxin,  is  produced.  In  each  case  there  is  an  immediate  fall 


30 
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FIG.  65.  —  EFFECT  OF  A  SINGLE  INJECTION  OF  TUBERCULIN,  SHOWING  THE 
"FALSE  RISE."     (Wright.) 


of  variable  duration,  followed  by  a  rise  to  a  higher  level  than  the 
initial  one  —  in  other  words,  there  is  a  negative  followed  by  a 
positive  phase.  (In  some  cases  there  is  a  sharp  "  false  rise  "  of 
short  duration,  which  precedes  the  negative  phase,  a  phenomenon 
which,  as  far  as  I  am  aware,  has  not  been  found  with  the  un- 
doubted antibodies.)  Now  this  rise,  as  has  been  already  pointed 
out,  is  to  some  extent  at  least  a  specific  one  ;  an  injection  of 
uterculin  does  not  cause  a  rise  in  the  opsonic  index  to  staphy- 


PHAGOCYTOSIS 


275 


lococci  or  pneumococci.  There  is,  therefore,  one  important  feature 
possessed  by  the  opsonins  in  common  with  the  antibodies  :  in  each 
case  an  injection  of  the  specific  antigen  causes  first  a  diminution 
and  then  an  increase  in  the  amount  present. 

There  is,  however,  an  important  difference.  In  the  other  anti- 
bodies— e.g.,  in  diphtheria  antitoxin — the  amount  present  in  the 
blood  can  be  raised  to  a  point  enormously  above  that  of  normal 
blood  by  a  series  of  inoculations  of  suitable  doses  of  toxin  at  suit- 
able intervals.  Here  the  effect  of  repeated  injections  is  a  cumu- 
lative one,  the  second  raising  the  index  above  the  level  which  it 


FIG.  66. — RESULT  OF  A  SINGLE  DOSE  OF  STAPHYLOCOCCIC  VACCINE, 
SHOWING  NEGATIVE  PHASE.     (Original.) 

reaches  after  the  first,  and  so  on.  But  in  the  case  of  the  opsonins 
to  most  bacteria  there  is  no  such  summation  of  results.  An  in- 
jection of  tuberculin  may  raise  the  opsonic  index  from  0-5  to  2  or 
a  little  higher,  but  with  a  second  injection  it  is  not  possible  to 
start  with  2  as  a  base  and  raise  the  index  to  3,  and  so  on.  The 
maximum  indices  are  not  very  much  above  the  normal  level.  In 
the  case  of  tubercle  it  is  very  unusual  to  find  an  index  as  high  as 
2,  whilst  with  the  organisms  of  suppuration,  etc.,  slightly  higher 
figures  may  occasionally  be  found.1  As  we  have  already  shown, 
this  does  not  prove  that  the  amount  of  tubercle  opsonin  present 

J  The  highest  indices  of  all  are  met  with  in  the  case  of  the  meningococcus. 
I  have  seen  them  exceed  10  in  patients  treated  with  vaccine,  and  higher 
figures  have  been  recorded.  The  explanation  of  these  figures  will  be  given 
subsequently. 

1 8— 2 


276      DIFFERENCE    BETWEEN    OPSONINS    AND    ANTIBODIES 

in  blood  never  exceeds  twice  the  normal — and  the  actual  amount 
may  be  much  more — but  anything  like  the  enormous  amounts 
which  can  be  obtained  when  working  with  antitoxins  or  agglutinins 
are  never  met  with  in  the  case  of  these  bacteria  at  least. 


FIG.  67.— SHOWING  THE  DIFFERENCE  BETWEEN  THE  BEHAVIOUR  OF  THE 
TRUE  ANTIBODIES  (DOTTED  LINE)  AND  OPSONIN  TO  SUCH  ORGANISMS  AS 
TUBERCLE  (LOWER  LINE)  WHEN  SUCCESSIVE  INJECTIONS  ARE  GIVEN. 
(Schematic.) 


FIG.  68. — SUMMATION  OF  NEGATIVE  PHASES  IN  OPSONIN  FORMATION  AS  THE 
RESULT  OF  INJECTIONS  IN  RAPID  SUCCESSION.     (Schematic  ) 

When  injections  are  repeated  during  the  negative  phase  a 
phenomenon  of  summation  may  be  met  with,  as  Wright  first 
pointed  out.  Here  the  first  injection  may  lower  the  index,  and 
the  second  and  third  lower  it  still  more,  until  a  very  low  figure  is 
reached.  A  phenomenon  similar  to  this  may  be  seen  after  the 
injections  of  toxins  (Fig.  68). 


PHAGOCYTOSIS  277 

It  is  on  these  facts  that  Wright's  vaccine  therapy,  or,  as  it  is 
sometimes  called,  opsonin  therapy,  is  based.  The  object  of  the 
treatment  is  to  bring  about  an  immunization  of  the  patient  by 
means  of  an  increase  of  the  opsonin  circulating  in  his  blood,  and 
this  is  achieved  by  the  injection  of  a  suitable  vaccine.  This  con- 
sists in  all  cases  of  the  dead  bodies  of  the  bacteria  causing  the 
disease.  In  the  case  of  tubercle  Koch's  new  tuberculin  (TR 
or  TE)  is  used  in  variable  amount,  but  not  usually  more  than 
T^5-  milligramme  of  dry  material  per  dose.  In  the  case  of  other 
bacteria  the  vaccine  is  prepared  by  cultivating  the  organism  on  a 
suitable  solid  culture  medium,  emulsifying  with  normal  saline 
solution,  and  heating  to  a  temperature  just  sufficient  to  insure 
sterility — usually  60°  C.  for  one  hour  is  requisite.  The  emulsion  is 
then  inoculated  on  to  a  culture  medium,  incubated  in  order  to  test 
its  sterility,  and  the  number  of  bacteria  which  it  contains  is  counted, 
in  order  to  determine  the  amount  to  be  used  as  a  dose.  Suitable 
dilutions  are  then  made.  The  dose  varies  with  different  bacteria. 
Thus,  with  staphylococci  250,000,000  to  1,000,000,000  cocci  may 
be  given,  whereas  with  B.  coli  25,000,000  is  usually  enough  for 
the  first  dose. 

The  treatment  is  controlled  by  a  frequent  estimation  of  the 
opsonic  index,  and  this  is  supposed  to  be  advisable  for  three 
reasons:  (i)  It  avoids  the  possibility  of  a  summation  of  the 
negative  phases,  and  so  a  worsening  of  the  patient's  condition  by 
lowering  his  immunity  to  the  infective  organism.  As  a  rule,  the 
negative  phase  is  but  of  short  duration,  but  occasionally  it  is  pro- 
longed, and  this  is  especially  the  case  when  large  doses  have  been 
given.  I  have  seen  it  as  long  as  three  weeks  in  a  case  of  tubercle. 
(2)  It  enables  a  suitable  dose  to  be  selected.  Thus,  if  we  find  a 
certain  number  of  bacteria  cause  a  long  negative  phase,  the  next 
injection  should  consist  of  a  smaller  one,  when  the  negative  phase 
may  be  reduced  and  the  rise  may  be  greater.  With  a  very  small 
dose  the  negative  phase  may  be  eliminated  altogether,  or  may  be 
reduced  so  much  that  it  is  overlooked.  (3)  Whilst  the  index  is 
raised  decidedly  above  normal  it  is  assumed  that  the  patient  is 
benefiting,  and  another  injection  is  only  required  when  it  begins 
to  fall.  As  a  rough  general  rule,  the  injections  have  to  be  repeated 
at  intervals  varying  from  a  week  or  fortnight,  but  individual 
patients  show  decided  differences  in  this  respect. 

Of  the  practical  success  of  this  treatment  in  certain  diseases 
there  can  be  no  doubt,  and  whatever  we  may  think  of  its  theoretical 


278  "  OPSONIN-THERAPY  " 

aspect,  Sir  Almroth  Wright  must  receive  the  greatest  credit  for 
its  introduction.  Before  his  researches  the  idea  of  injecting  a 
vaccine  into  a  patient  already  suffering  from  a  bacterial  disease 
was  unthought  of,  although,  of  course,  it  was  well  known  as  a 
method  of  producing  immunity  when  disease  was  feared.  The 
question  is  often  asked,  Why  inject  more  staphylococci  into  a 
patient  who  has  already  too  many  ?  The  answer  may,  perhaps, 
be  as  follows  :  The  staphylococci  which  cause  the  lesion  come 
into  contact  with  dead  and  diseased  tissues  only,  and  it  is  easily 
conceivable  that  these  may  be  very  unsuitable  to  discharge  so 
vital  a  function  as  the  formation  of  antibodies,  whereas  a  few  cocci 
injected  into  the  healthy  tissues  may  cause  a  large  amount.  This, 
however,  does  not  explain  the  benefit  which  has  been  observed  in 
some  cases  of  endocarditis  and  other  haemal  infections,  for  in  them 
the  bacteria  must  be  constantly  gaining  access  to  the  healthy 
endothelial  cells,  if  to  no  others.  But  it  is  well  known  that  not  all 
the  tissues  are  equally  adapted  for  the  production  of  antibodies ; 
thus,  when  diphtheria  toxin  is  injected  into  the  blood-stream  little, 
if  any,  production  of  antitoxin  takes  place.  As  a  general  rule, 
when  antibodies  are  required  the  blood-stream  is  the  worst  place 
in  which  to  inject  the  antigen,  the  serous  membranes  next,  and 
the  connective  tissues  the  best.  Dr.  Whitfield  has  suggested  to 
me  that  the  reason  may  be  that  the  stimulation  of  the  opsonins 
occurs  best  when  dead  bacteria  are  injected.  Thus  in  the  early 
stage  of  the  disease  only  living  organisms  are  present,  whilst  later 
we  must  suppose  some  are  killed  or  die  from  some  cause,  and 
then  the  stimulation  of  opsonin  formation  begins.  The  idea  is 
worth  considering,  but  the  subject  is  still  obscure. 

As  regards  the  nature  of  these  results  :  In  tubercle,  speaking 
from  my  own  experience,  I  can  only  report  a  moderate  degree  of 
success,  and  this  only  in  small  lesions,  such  as  tubercle  of  the  iris 
or  cornea  and  of  tuberculous  ulcers.  I  have  had  but  one  or  two 
encouraging  results  and  numerous  failures  with  tuberculous  glands, 
bone  disease,  etc.,  though  others  have  apparently  been  more 
successful.  In  phthisis  there  appears  to  be  some  slight  benefit 
when  combined  with  other  treatment,  and  tuberculous  sinuses 
sometimes  heal  very  quickly.  I  should  only  recommend  the 
treatment  myself  as  an  adjunct  toother  methods,  or  when  surgical 
interference  is  impossible  or  inadvisable. 

With  the  diseases  due  to  acute  infections  with  staphylococci, 
pneumococci,  B.  coli,  and  some  other  organisms,  however,  the 


PHAGOCYTOSIS  279 

results  are  most  beneficial.  We  may  often  see  boils  apparently 
on  the  point  of  bursting  retrocede  in  a  most  striking  manner  after 
a  single  injection  of  staphylococcic  vaccine,  and  pustular  acne  is 
often  equally  benefited.  Localized  lesions  of  pneumococcic  origin 
often  clear  up  quickly  under  the  action  of  pneumococcic  vaccine  : 
thus  a  case  of  empyema  of  the  frontal  sinus,  due  to  this  origin  and 
of  four  years'  duration,  was  cured  in  five  injections,  spread  over  a 
period  of  about  twqjnonths.  Numerous  cases  of  cure  of  chronic 
infections  of  the  urinary  tract  with  B.  coli  have  been  recorded,  and 
some  cases  of  gonorrhceal  arthritis  have  been  cured  in  a  remark- 
able manner.  In  a  case  of  my  own  a  patient,  with  five  large  and 
numerous  small  joints  affected,  was  completely  cured  in  three 
months,  after  having  been  crippled  for  over  two  years.  One  or  two 
undoubted  cases  of  ulcerative  endocarditis  have  been  cured,  and 
others  in  which  there  was  a  haemic  infection  (with  streptococci), 
though  the  evidence  in  favour  of  a  valvular  infection  is  less  con- 
vincing. The  results  in  cases  of  Malta  fever  are  also  very 
encouraging.  As  a  rule,  however,  we  may  say  that  the  special 
scope  of  the  method  is  in  the  treatment  of  localized  infections. 

A  point  of  great  practical  importance,  and  one  that  has  some 
theoretical  interest  as  pointing  to  a  high  degree  of  specificity  in 
the  opsonins,  is  the  fact  that  good  results  are  sometimes  obtained 
only  when  the  vaccine  used  is  from  a  culture  of  the  organism  in 
question  from  the  patient  himself.  This  is  sometimes  seen  in 
staphylococcic  infections ;  acne  is  occasionally  very  resistant  to 
stock  vaccines,  and  yields  readily  to  treatment  with  an  emulsion 
prepared  from  a  culture  from  the  patient's  own  pus.  This 
phenomenon  is  specially  marked  in  the  case  of  streptococci  and 
B.  coli. 

In  admitting  the  success  of  vaccine  therapy,  we  do  not  neces- 
sarily admit  the  truth  of  the  theory  on  which  it  is  based,  nor  the 
necessity  for  the  opsonic  control  of  the  doses.  It  is  certainly  true 
in  general  that  with  acute  lesions  there  is  a  low  opsonic  index, 
and  that  when  amelioration  or  cure  takes  place  a  rise  to  or 
above  normal  occurs,  but  this  is  not  invariably  the  case.  Thus, 
occasionally  tuberculous  patients  improve  whilst  the  index 
remains  low,  and  those  with  a  meningococcal  infection  often  go 
steadily  downhill  whilst  the  index  is  very  high,  though  in  the 
latter  case  the  symptoms  are  in  general  more  severe  when  the 
index  falls.  Now  it  is  quite  true  and  perfectly  conceivable  that 
the  continued  existence  of  a  lesion  in  spite  of  a  very  high  opsonic 


280  OBJECTIONS   TO   THE    OPSONIN    THEORY 

index  may  be  due  to  a  failure  of  the  serum  or  leucocytes  to  gain 
access  to  the  lesions  which  are  densely  surrounded  by  inflam- 
matory material.  .  We  have  adduced  a  similar  reason  to  explain 
the  non-success  of  certain  bactericidal  sera.  But  it  is  otherwise 
when  we  find  that  a  patient  improves  when  there  is  a  low  index, 
for  here  we  must  admit  that  even  this  deficient  amount  of  opsonin 
is  sufficient ;  and,  further,  it  is  impossible  to  explain  the  formation 
of-  new  lesions  (e.g.,  staphylococcic)  in  patients  in  whom  the 
opsonic  index  is  high — often  very  high — on  any  such  grounds. 
Again,  a  great  rise  in  the  opsonic  index  not  infrequently  occurs 
just  before  death,  as  in  the  chart  of  the  index  in  a  fatal  case  of 
erysipelas  already  figured.  The  more  carefully  the  opsonic  index 
is  considered,  the  more  certain  will  it  appear  that  a  high  index  is 
not  an  indication  of  immunity  ;  it  neither  proves  that  the  lesion 
is  undergoing  cure  nor  that  a  fresh  infection  will  not  occur.  It 
may,  of  course,  occur  concurrently  with  other  properties  in  the 
blood  or  tissues  on  which  immunity  does  depend — indeed,  since  it 
is  commonly  due  to  the  presence  of  a  natural  or  artificial  vaccine, 
it  usually  does  so — but  the  parallelism  between  a  rise  in  the 
amount  of  opsonins  and  an  increase  in  the  grade  of  immunity  is 
not  absolute.  -Nor  is  a  low  index  any  proof  of  lack  of  immunity, 
since  patients  may  improve  remarkably  during  a  prolonged 
negative  phase.  One  of  the  most  striking  cases  of  amelioration 
of  a  severe  case  of  tubercle  which  I  have  ever  seen  occurred 
during  a  negative  phase  lasting  over  three  weeks.  Allen  has 
noted  a  similar  occurrence  in  gonorrhreal  infections,  from  which, 
however,  he  draws  the  assumption  that  the  clinical  signs  are 
a  totally  unreliable  guide  to  the  appropriate  time  for  a  fresh 
injection — a  deduction  which  is  logical  only  if  we  regard  the 
raising  of  the  opsonic  index,  and  not  the  cure  of  the  patient,  as 
the  object  of  treatment. 

It  seems  probable,  from  a  consideration  of  the  phenomena  of 
phagocytosis  in  vitro,  that  a  very  small  amount  of  opsonin — even 
less  than  that  which  is  present  in  a  serum  in  which  the  index  is 
very  low — is  quite  sufficient  to  sensitize  any  bacteria  that  are 
likely  to  gain  access  to  the  tissues  or  blood.  In  our  laboratory 
experiments  the  conditions  are  certainly  much  less  favourable 
than  they  are  in  the  living  body  ;  the  leucocytes  are  certainly  not 
in  the  same  state  of  functional  activity  as  they  are  in  the  body, 
and  there  is  a  limited  supply  of  serum  instead  of  a  constant  stream 
thereof.  In  spite  of  this,  an  enormous  number  of  bacteria  are 


PHAGOCYTOSIS  28l 

taken  up,  and  in  some  cases  digested,  within  a  few  minutes.  In 
the  body,  of  course,  the  action  may  go  on  for  hours.  The  opsonin- 
leucocyte  mechanism  would  appear  far  stronger  than  is  necessary 
for  the  defence  of  the  body.  That  it  is  not  so  indicates  some 
fallacy  in  the  conclusions  to  be  derived  from  these  experiments 
in  vitro.  We  shall  revert  to  this  subject  subsequently,  and  in  the 
meantime  be  content  with  pointing  out  that  where  a  very  small 
amount  of  opsonin  would  appear  sufficient  for  the  resources  of 
the  body,  but  little  importance  can  be  attached  to  small  fluctua- 
tions, or  to  a  rise,  e.g.,  from  0-8  to  i. 

The  dread  of  a  low  opsonic  index  appears  to  have  arisen  on 
purely  theoretical  grounds,  and  the  only  direct  research  on  the 
subject  which  seems  to  have  been  undertaken  points  rather  in  the 
other  direction.  According  to  Pfeiffer  and  Friedberger,  guinea- 
pigs  injected  with  bacterial  vaccines  (typhoid  and  cholera)  do  not 
thereby  become  hypersensitive  to  doses  of  living  cultures  given 
twelve  or  thirty-six  hours  afterwards  ;  on  the  contrary,  they  have 
acquired  an  increased  power  of  resistance,  even  after  the  shorter 
period.  And  a  very  remarkable  fact  was  noticed  :  this  increased 
resistance  was  not  specific,  since  animals  injected  with  heated 
typhoid  bacilli  survived  a  lethal  dose  of  cholera  as  well  as  of 
typhoid.  They  conclude  that  the  fear  of  a  negative  phase  is 
exaggerated  ;  and  it  must  not  be  forgotten  that  the  essence  of 
the  "  opsonin  therapy  "  consists  in  administering  a  dose  of  vaccine, 
in  the  first  instance,  while  the  index  is  low. 

There  is  thus  no  direct  proof  that  the  period  of  the  negative 
phase  is  coincident  with  the  period  of  hypersensitiveness  to 
infection.  And  when  we  compare  it  with  the  period  of  increased 
sensitiveness  to  toxins,  we  find  that,  whereas  the  negative  phase 
comes  on  almost  immediately,  the  hypersensitiveness  to  toxins 
or  tuberculin,  or  anaphylaxis  to  serum,  takes  some  days  to 
develop. 

Other  theoretical  interpretations  of  the  undoubted  good  effects 
of  vaccine  therapy  are  possible.  Thus,  a  very  probable  explana- 
tion is  that  it  causes  a  local  reaction  in  the  form  of  an  aseptic 
inflammatory  process  in  the  neighbourhood  of  the  lesion,  which, 
like  the  similar  reaction  caused  by  ultra-violet  or  X  rays,  has  (in 
some  way  not  yet  understood)  a  curative  effect.  The  nature  of 
these  "  reactions  "  is  considered  subsequently ;  in  the  meantime 
it  is  sufficient  to  say  that  in  the  case  of  tubercle  (and  it  is 
probably  a  general  effect)  an  injection  of  dead  bacilli,  or  of  the 


282  ALTERNATIVE  EXPLANATIONS 

products  thereof,  causes  a  sharp  rise  in  temperature  and  an 
inflammatory  process  around  the  tuberculous  focus.  If  the  dose 
of  tuberculin  be  greatly  reduced  the  local  reaction  takes  place, 
but  there  is  no  rise  of  temperature.  This  is  best  seen  when 
small  doses  of  TR  are  used  in  the  treatment  of  tuberculous 
iritis,  in  which  the  iris  can  often  be  seen  to  become  injected  after 
each  dose  ;  and  I  have  observed  the  same  reaction  in  a  very 
marked  form  after  the  use  of  diluted  old  tuberculin  in  von 
Pirquet's  reaction.  In  this  case  the  dose  absorbed  must  have 
been  infinitesimal,  since  the  temperature  did  not  show  the  slightest 
sign  of  a  rise. 

Other  possibilities  are  that  the  vaccine  may  cause  a  general 
tissue  immunity,  or  that  it  may  produce  some  degree  of  immunity 
on  the  part  of  the  leucocytes,  or  may  at  least  alter  them  in  some 
way  so  that  they  are  more  able  to  perform  their  duties  as  phago- 
cytes ;  and,  of  course,  other  antibodies,  such  as  antiendotoxins, 
may  be  produced  as  a  result  of  the  injection,  and  of  these  the 
opsonic  index  affords  us  no  estimate. 

In  reverting  to  the  question  of  the  nature  and  properties  of  the 
opsomns,  the  question  of  their  thermo-stability  first  claims  our 
attention.  The  results  obtained  by  various  observers  are  not 
quite  in  accord,  and  indicate  very  clearly  that  more  than  one 
substance  may  have  the  same  action.  The  opsonin  present  in 
normal  serum  is  in  a  high  degree  thermolabile.  It  is  destroyed 
by  heating  to  55°  C.  for  half  to  one  hour  ;  at  60°  C.  most  disappears 
in  five  minutes,  the  rest  more  slowly,  little  being  left  in  fifteen 
minutes.  Wright  and  Reid,  however,  found  that  in  cases  of 
tuberculosis  some  of  the  opsonin  is  more  thermostable,  and 
whereas  in  heating  a  normal  control  to  60°  C.  for  ten  minutes 
reduces  the  opsonic  index  to  almost  nothing,  the  same  proceeding 
may  only  lower  the  index  of  a  tuberculous  serum  to  0-4  or  so, 
though  the  indices  of  both  samples  were  formerly  the  same. 
They  suggested  this  as  a  means  for  the  diagnosis  of  tubercle. 
Other  observers  have  failed  to  corroborate  their  results,  and  they 
are  certainly  not  true  of  all  cases.  Dean  showed  that  in  certain 
sera  obtained  by  the  high  immunization  of  animals  to  certain 
bacteria  (staphylococci,  dysentery,  and  typhoid  bacilli)  there  are 
substances  which  act  as  opsonins,  and  which  are  thermostable. 
His  results  have  been  corroborated  for  pneumococcic  serum  by 
Macdonald  and  Rosenau,  by  Muir  and  Martin,  and  many  others. 
It  is  evident,  therefore,  that  there  is  more  than  one  substance 


PHAGOCYTOSIS  283 

which  can  prepare  bacteria  for  phagocytosis.  There  is  a  thermo- 
labile  substance  which  occurs  in  normal  serum,  and  a  thermo- 
stable one  which  is  found  in  immune  serum  ;  and  this  latter  also 
contains  a  thermolabile  substance,  since  (as  a  rule)  its  index  is 
lowered  by  heat.  Thermostable  opsonin  occurs  in  minute  traces 
in  normal  serum,  since  the  index  is  never  reduced  quite  to  the 
level  seen  in  a  control  specimen  made  with  normal  saline  by 
heating  to  60°  C  ,  and  we  need  have  no  hesitation  in  recognizing  it 
as  a  specific  antibody.  It  will  be  convenient  to  deal  with  it  first, 
and  the  question  naturally  arises,  Is  it  amboceptor  ?  In  other 
words,  Has  amboceptor  the  power  of  preparing  bacteria  for 
phagocytosis  in  addition  to  sensitizing  them  to  the  action  of 
complement  ?  The  two  substances  arise  under  the  same  con- 
ditions, and  are  identical  in  their  power  of  resisting  heat,  faculty 
of  combining  with  bacteria,  and  in  their  specificity.  The  second 
question  arises,  Assuming  thermostable  opsonin  is  amboceptor,  is 
the  action  of  complement  also  useful  in  preparing  bacteria  for 
phagocytosis,  or  does  the  process  go  on  equally  well  without  it  ? 
Now  it  is  certain  that  complement  is  not  necessary  for  the  action 
of  thermostable  opsonin;  otherwise  it  would  only  exert  its  action 
in  a  heated  serum  when  subsequently  activated  by  fresh  serum, 
and  this  is  not  the  case.  If  thermostable  opsonin  is  amboceptor, 
therefore,  it  can  exert  its  effects  without  the  action  of  com- 
plement. But  some  experiments  go  to  show  that  thermostable 
opsonin  may  be  more  potent  when  reactivated.  Thus  Crofton 
found  an  antistreptococcic  serum  might  stimulate  phagocytosis 
more  when  mixed  with  fresh  human  serum  than  with  an  equal 
amount  of  normal  saline. 

Similar  results  have  been  obtained  more  recently  by  Dean, 
who  finds  that  the  opsonic  effect  obtained  by  heated  serum  and 
normal  serum  may  be  greater  than  the  sum  of  the  two  effects 
separately.  The  subject  has  been  very  carefully  investigated  by 
Chapin  and  Cowie,  who  were  able  to  avoid  the  possibility  of 
certain  errors  by  performing  their  saturation  experiments  in  a 
cold  room,  kept  at  o°  C.  throughout  the  experiment.  They  found 
that  a  normal  human  serum  treated  with  staphylococci  at  this 
temperature  might  have  the  whole  of  its  opsonic  power  removed, 
and  yet  would  still  reactivate  a  heated  serum — i.e.,  the  thermo- 
stable opsonin  combines  with  bacteria  at  o°  C.,  and  is  probably 
amboceptor.  They  found  that  staphylococci  treated  with  normal 
serum  at  o°  C.  and  then  washed  are  slightly  more  susceptible  to 


284  AMBOCEPTOR   AND    OPSONIN 

phagocytosis  than  are  normal  ones,  but  the  difference  is  not  great. 
They  are,  however,  much  more  easily  opsonized  by  normal  serum, 
or  by  serum  that  has  had  its  amboceptor  removed  by  treatment 
with  staphylococci  in  the  cold. 

In  other  cases  the  conditions  are  more  complex,  for  when  a 
potent  bacteriolytic  serum  is  present,  bacteriolysis  may  occur  to 
such  an  extent  as  to  diminish .  the  number  of  organisms  which 
can  be  taken  up  by  the  leucocytes.  We  then  get  the  "  reversed 
ratio  "  phenomenon  described  by  Leishman  and  Dean.  It  is  as 
follows :  Under  ordinary  conditions  the  index  falls  greatly  on 
heating,  as  has  been  shown.  This  is  called  the  normal  ratio. 
But  in  some  of  the  potent  sera  obtained  from  highly  immunized 
animals  the  opsoni^  index  may  apparently  rise  after  heating  to 
two  or  three  times  that  of  the  raw  serum.  This  Dean  explains 
— and  his  explanation  is  an  extremely  rational  one — by  invoking 
the  bacteriolytic  action  of  the  unheated  serum.  The  number  of 
bacteria  in  the  emulsion  is  reduced,  so  that  there  are  fewer  for  the 
leucocyte  to  take  up ;  some  that  are  not  completely  dissolved 
may  lose  their  power  of  retaining  stains  and  become  invisible ; 
bacteria  partially  acted  on  may  be  readily  digested  within  the 
leucocyte,  so  that  they  are  not  counted  ;  and,  lastly,  the  dissolved 
bacteria  may  have  a  toxic  effect  on  the  leucocytes.  The 
phenomenon  of  the  reversed  ratio  may  be  taken  as  an  argument 
in  favour  of  the  equivalence  of  thermostable  opsonin  and 
amboceptor. 

The  strongest  argument,  however,  is  derived  from  the  experi- 
ments of  Dean,  who  has  shown  that  in  different  samples  of 
immune  sera  there  is  a  distinct  parallelism  between  the  two 
functions  :  when  the  serum  is  powerful  as  a  bacteriolytic  agent, 
when  activated  with  a  suitable  complement,  it  is  also  powerful  as 
an  opsonin  after  heating.  It  must  be  admitted,  of  course,  that  a 
serum  may  be  opsonic,  but  not  bacteriolytic  ;  but  this  is  explicable 
on  the  assumption  that  much  less  of  the  substance  is  required  to 
sensitize  the  bacterium  to  the  attack  of  leucocytes  than  is  necessary 
to  render  it  soluble  by  complement.  This  has  been  confirmed 
by  Neufeld  and  Bickel,  who  found  that  a  very  minute  amount  of 
haemolytic  serum,  far  less  than  would  produce  haemolysis,  would 
act  as  a  haemopsonin. 

The  opsonic  index  does  not  rise  pan  passu  with  the  bacteriolytic 
power,  but  this  is  partly  due  to  the  fact  that  the  criteria  are 


PHAGOCYTOSIS  285 

different  in  the  two  cases.  We  have  already  shown  that  incre- 
ments in  the  amounts  of  opsonin  cause  smaller  and  smaller 
rises  in  the  opsonic  index  as  we  proceed.  There  is,  however, 
a  much  closer  parallelism  between  the  bacteriolytic  power  and 
the  amount  of  thermostable  opsonin  present  as  shown  by  the 
degree  of  dilution.  This  is  well  shown  (in  the  case  of  typhoid 
fever)  by  the  chart  given  by  Klien  and  inserted  previously 

(Fig.  58). 

To  sum  up :  Amboceptor  appears  to  have  the  power  of 
sensitizing  bacteria  for  phagocytosis,  and  this  power  appears  to 
be  increased  by  the  concurrent  action  of  complement.  Further, 
there  appears  to  be  no  sufficient  evidence  for  the  existence  of 
a  thermostable  opsonin  apart  from  amboceptor,  as  has  been 
maintained  by  Neufeld  and  Hime. 

(There  is  an  additional  possibility  that  the  part  of  a  thermo- 
stable opsonin  may  be  enacted  by  agglutinin. 

I  believe  that  in  the  case  of  the  haemopsonins  of  normal 
human  serum  the  substance  is  a  thermostable  agglutinin  with  a 
second  thermolabile  zymotoxic  group.  Natural  haemopsonic  sera 
are,  as  far  as  I  have  seen,  always  powerful  agglutinators  of  the 
red  corpuscles  which  they  opsonize,  and  when  they  are  heated  to 
60°  C.  the  opsonic  power  is  destroyed,  but  the  agglutinative 
faculty  is  unaltered.) 

These  facts  may  serve  to  explain  the  discordant  results  as  to 
the  presence  of  thermostable  opsonins  in  the  sera  of  tuberculous 
patients.  It  has  been  shown  by  Bruck  that  antibodies  to  the 
tubercle  bacillus  are  not  always  or  usually  present  in  the  blood  of 
infected  persons,  and  it  is  only  when  they  are  present  that  we 
should  find  a  thermostable  opsonin. 

If  thermostable  opsonins  resemble  amboceptor  in  their  pro- 
perties, there  is  an  equally  close  resemblance  between  thermo- 
labile opsonin  and  complement.  Each  occurs  in  normal  serum, 
and  is  destroyed  by  a  short  heating  to  55°  to  60°  C.  Are  they  the 
same  ? 

The  main  fact  against  the  theory  of  their  identity  is  the 
specificity,  partial  though  it  may  be,  of  the  opsonins  ;  for  there  is 
no  reason  to  think  that  different  bacteria  are  attacked  by  different 
complements,  even  if  we  accept  the  theory  of  the  multiplicity  of 
these  bodies  to  the  fullest  degree.  But  we  have  already  seen 
that  the  specificity  of  the  opsonins  is  not  complete,  and  that  the 


286  COMPLEMENT    AND    OPSONIN 

whole    of   the   staphylococcic  opsonin  may    be    removed   by   the 
addition  of  sufficient  amounts  of  tubercle  bacilli.1 

A  second  fact,  closely  allied  and  perhaps  in  reality  identical 
with  the  foregoing,  is  the  rise  in  a  particular  opsonin  after  an 
injection  of  a  suitable  vaccine,  the  others  remaining  constant. 
This  rise  cannot  be  accounted  for  (in  my  opinion,  at  least)  by 
the  appearance  of  small  quantities  of  thermostable  opsonin, 
since  it  may  occur  when  this  substance  cannot  be  found  in  the 
serum. 

On  the  other  hand,  there  are  very  remarkable  analogies  between 
the  two  substances.  In  each  there  is  the  same  difference  of 
opinion  as  to  whether  it  occurs  in  normal  plasma,  or  is  only 
developed  when  clotting  and  destruction  of  leucocytes  occur. 
Wright  and  Douglas  found  the  amount  of  opsonin  present  in 
serum  and  in  citrated  plasma  exactly  the  same,  whereas  Briscoe 
found  that  very  little  phagocytosis  took  place  when  staphylococci 
were  injected  into  a  surviving  heart  in  which  no  clotting  took 
place.  These  divergencies  are  quite  similar  to  those  found  by 
different  investigators  in  the  case  of  complement. 

Again,  it  has  been  already  shown  that  when  a  blood-clot 
contracts,  the  first  serum  which  can  be  collected  is  poor  in  com- 
plement compared  with  that  which  follows,  and  that  after  a  time 
the  amount  again  diminishes.  An  exactly  similar  phenomenon 
may  sometimes,  though  apparently  not  always,  be  demonstrated 
with  opsonin  (Henderson  Smith).  Hence  an  important  practical 
point :  the  patient's  blood  should  always  be  collected  at  the  same 
time  as  the  control  in  determinations  of  the  opsonic  index. 

Thirdly,  it  has  been  shown  by  Levaditi  that  the  aqueous 
humour  of  the  rabbit  contains  no  complement  and  but  a  trace  of 
opsonin.  But  when  the  fluid  which  recollects  after  puncture  was 
examined,  it  was  found  to  be  rich  in  both  substances.  He  found 
a  similar  relation  between  the  two  substances  in  redema  fluid. 
As  against  these  results  we  have  to  put  the  researches  of  Leding- 

1  Since  the  above  was  written  Muir  and  Browning  have  adduced  very  definite 
evidence  of  a  partial  specificity  in  the  case  of  the  complements.  They  find 
that  the  bactericidal  action  of  normal  serum  may  be  due  to  the  direct  action 
of  complements,  and  that,  on  weakening  normal  serum  by  successive  additions 
of  dead  bacteria,  the  first  effect  is  a  falling  off  in  the  bactericidal  action  as 
tested  on  that  bacterium.  Then  the  bactericidal  action  on  the  other  bacteria  is 
diminished,  and  with  a  larger  addition  the  haemolytic  complement  is  absorbed. 
This  indicates  features  exactly  like  the  partial  specificity  seen  in  opsonins, 
and  a  similar  absorption  without  the  intervention  of  an  immune  body. 


PHAGOCYTOSIS  287 

ham  and  Bulloch,  who  found  that  when  the  number  of  leucocytes 
in  the  blood  was  increased  by  injections  of  cinnamate  of  sodium, 
there  was  an  increase  in  the  complement,  but  not  in  the 
opsonin. 

It  may  be  pointed  out  that  if  opsonin  and  complement  are  the 
same,  we  must  suppose  that  the  opsonin  test  is  the  most  delicate 
method  of  demonstrating  this  substance  that  we  have,  since 
phagocytosis  may  be  facilitated  by  substancesfwnicnjji}  comple- 
ments cannot  be  detected  by  ordinary  tests.  Further,  we  must 
assume  that  it  unites  with  bacteria  direct,  and  sensitizes  them  for 
phagocytosis  without  the  intervention  of  amboceptor.  There  is 
no  serious  difficulty  in  accepting  both  suppositions. 

Lastly,  Muir  has  shown  that  the  substances  which  have  the 
power  of  absorbing  complement  (such  as  compounds  of  red  blood- 
corpuscles  and  their  amboceptor)  also  remove  the  thermolabile 
opsonin.  We  are  forced,  therefore,  to  the  conclusion  that  com- 
plements may  play  the  part  of  opsonins.  But  to  do  this  we  must 
necessarily  broaden  our  ideas  of  the  complements,  and  attribute 
to  them  some  degree  of  specificity  ;  otherwise  the  opsonic  index  of 
any  given  sample  of  serum  as  measured  against  a  given  control 
should  be  always  the  same,  which,  as  we  have  already  emphasized, 
is  not  the  case  (see  footnote,  p.  286). 

These  results  suggest  another  train  of  ideas  as  to  the  role  of 
bacteriolysis  in  immunity.  We  have  already  seen  reason  to 
believe  that  this  is  not  of  the  greatest  importance,  and  have  found 
it  difficult  to  think  that  so  elaborate  a  mechanism  should  be  of  so 
little  apparent  use.  May  it  not  be  that  the  complements  are 
specially  intended  for  use  as  opsonins,  and  that  their  action  in 
bacteriolysis  is  a  secondary  one,  and  comparatively  of  less 
importance  ?  This,  of  course,  is  a  close  approximation  to  Metch- 
nikoff's  views,  but  there  is  this  difference :  his  cytase  is  a 
digestive  ferment  which,  in  the  case  of  microcytase,  is  adapted  to 
attack  all  sorts  of  bacterial  proteid.  But  with  the  opsonins  or 
complements  we  must  assume  that  different  molecules  occur 
which  have  different  combining  affinities  for  the  protoplasm  of 
different  bacteria,  or,  in  other  words,  which  differ  slightly  in  their 
haptophore  groups.  Yet  this  difference  is  one  in  degree  and  not 
in  kind,  for  they  all  have  some  power  of  uniting  with  all  bacteria, 
and  a  great  power  of  uniting  with  the  bacterium-amboceptor 
combination. 

On  this  theory  the  appearance  of  amboceptor  will  take  on  a  new 


288  SOURCE    OF    OPSONIN 

significance,  and  we  must  regard  this  substance  as  a  device  for 
attaching  more  complements  and  more  varieties  of  complement  to 
an  invading  bacteria  than  can  easily  combine  with  it  direct.  In 
other  words,  we  must  regard  the  cytophile  group  of  the  ambo- 
ceptor  as  being  specific,  whilst  the  complementophile  group  has 
the  modified  specificity  which  we  attribute  to  the  opsonins.  The 
presence  of  amboceptor  will  therefore  enable  the  bacterium  to  be 
prepared  for  phagocytosis  by  the  concurrent  action  of  many  com- 
plements which  otherwise  would  only  be  able  to  attack  it  with 
great  difficulty. 

And  many  facts,  notably  the  liberation  of  endotoxin  taking 
place  when  bacteriolysis  occurs,  would  lead  us  to  believe  that 
this  preparation  for  phagocytosis  is  the  true  function  of  ambo- 
ceptor and  complements,  and  that  the  appearance  of  the  latter  in 
excess  is  a  comparatively  rare  phenomenon  in  disease,  and  when 
it  occurs  in  enormous  amounts  (such  as  is  seen  in  highly  immunized 
animals)  is  an  artificial  phenomenon  comparable  with  the  enormous 
amounts  of  antitoxin  seen  in  antitoxin -horses.  Recovery  from  an 
attack  of  disease  caused  by  B.  coli  may  occur  without  the  appear- 
ance of  any  amboceptor  to  B.  coli  demonstrable  by  ordinary  tests  ; 
there  may,  nevertheless,  be  quite  sufficient  to  act  as  a  thermo- 
stable opsonin.  We  are  far  from  denying  that  bacteriolysis  ever 
occurs  under  natural  conditions,  but  when  there  are  plenty  of 
leucocytes  of  sufficient  functional  activity,  it  is  difficult  to  avoid 
the  conclusion  that  they  would  ingest  the  bacteria  when  these 
were  sensitized  by  complement  alone,  or  complement  and  a  little 
amboceptor,  and  before  this  latter  substance  had  been  developed 
in  amount  sufficient  to  cause  bacteriolysis.  This  latter  process 
may  perhaps  be  the  last  line  of  defence,  to  be  used  only  if  the 
leucocytes  are  injured  by  the  toxins  or  by  the  high  temperature, 
or  if  they  are  present  in  insufficient  numbers. 

It  has  been  pointed  out  already  that  there  is  some  reason  to 
think  that,  whilst  complement  and  amboceptor  can  each  sensitize 
for  phagocytosis  separately,  they  exert  a  more  potent  action  when 
both  are  present. 

As  regards  the  source  of  opsonin,  little  is  definitely  known. 
If  we  regard  the  thermolable  opsonin  as  identical  with  comple- 
ment, we  shall  regard  it  as  probably  derived  from  the  polynuclear 
leucocytes,  and  this  is  corroborated  by  Levaditi's  observations 
on  the  aqueous  humour.  Eyre  has  also  shown  that  the  amount 
of  opsonin  (to  pneumococci)  in  the  serum  in  pneumonia  may  be 


PHAGOCYTOSIS 


289 


roughly  parallel  with  the  number  of  leucocytes  per  cubic   milli- 
metre (Fig.  69). 

This,  however,  was  not  corroborated  by  Bulloch  and  Leding- 
ham  in  the  case  of  the  hyperleucocytosis  caused  by  cinnamate 
of  soda.  But  it  is  highly  doubtful  whether  leucocytes  hurried 
prematurely  from  the  bone-marrow,  etc.,  are,  as  the  result  of  the 
injection  of  chemical  substances,  as  active  functionally  as  those 
occurring  normally  in  that  situation  ;  and  this  is  corroborated 


FIG.  69. — RELATION  BETWEEN  LEUCOCYTES,  OPSONIC  INDEX,  AND  TEMPERA- 
TURE IN  A  CASE  OF  PNEUMONIA.     (Eyre.) 

Dotted  line  =  number  of  leucocytes  per  cubic  millimetre  ;  thick  line  =  opsonic 
index;  thin  line  =  temperature. 

by  the  fact  that  these  observers  found  the  leucocytes  in  question 
deficient  in  phagocytic  powers.  The  point  is  one  of  some  im- 
portance in  connection  with  the  lack  of  benefit  which  so  often 
follows  an  artificial  leucocytosis  brought  about  for  therapeutic 
purposes. 

A  few  words  on  the  subject  of  MetchnikofFs  views  on  the  op- 
sonins  may  be  added.  He  thinks  that  when  bacteria  gain  access 
to  the  blood  or  tissues,  the  presence  of  opsonins  or  other  pre- 
paratory substances  is  unnecessary,  and  the  unaltered  organisms 
can  be  attacked  by  the  fresh  and  vigorous  leucocytes.  The 

19 


2QO  METCHNIKOFF  S   VIEWS    ON    OPSONINS 

absence  of  phagocytosis  in  vitro  in  the  absence  of  serum  he 
attributes  to  a  weakening  and  injury  of  the  cells,  due  to  the 
method  by  which  they  are  prepared,  and  admits  that  these 
weakened  and  altered  leucocytes  will  ingest  bacteria  more  easily 
and  more  quickly  if  the  latter  have  previously  been  prepared  by 
the  action  of  serum.  He  admits,  however,  that  the  opsonic  index 
determines  the  defensive  resources  of  the  blood,  and  in  doing  so 
would  appear  to  range  himself  definitely  amongst  the  adherents 
of  the  opsonin  theory.  But  there  is  no  reason  to  think  that 
washed  leucocytes  are  weakened  in  respect  of  their  phagocytic 
powers ;  they  can  take  up  enormous  numbers  of  (opsonized) 
bacteria  in  a  very  short  space  of  time,  and  it  is  difficult  to  believe 
that  they  could  take  up  more  in  the  living  body ;  and  if,  as  there 
is  reason  to  think,  phagocytosis  is  a  physical  process  akin  to 
agglutination,  the  functional  activity  of  the  leucocytes  is  a  factor 
of  little  importance  in  phagocytosis,  though  essential  for  the 
other,  equally  necessary,  phenomena  of  digestion  and  solution 
which  Jake  place  subsequently.  Metchnikoff  finds  that  washed 
Bacteria  can  take  up  large  numbers  of  bacteria  slowly,  even  in 
the  absence  of  serum.  This,  however,  proves  nothing,  since  we 
have  seen  there  is  some  reason  to  believe  that  opsonin  may  be 
formed  from  the  leucocytes  themselves.  But,  as  a  matter  of  fact, 
the  increase  in  phagocytosis  in  preparations  incubated  for  one  or 
two  hours  as  compared  with  those  incubated  for  fifteen  minutes 
is  slight  as  compared  with  that  consequent  on  the  addition  of 
serum. 

The  influence  of  the  source  of  the  leucocytes  taking  part  in 
phagocytosis  is  not  yet  fully  investigated,  and  there  are  no  facts 
known  at  present  which  tend  to  show  that  those  from  an  immunized 
animal  have  any  special  powers  in  this  direction.  Bulloch  showed 
in  a  few  cases  that  leucocytes  from  different  sources  would  take 
up  the  same  number  of  bacteria  if  used  with  the  same  opsonic 
sera.  There  are  also  observations  tending  to  show  that  diseased 
or  abnormal  leucocytes — e.g.,  those  produced  in  excess  as  a  result 
of  the  injection  of  certain  substances,  such  as  nuclein — are  deficient 
in  phagocytic  activity. 

In  a  very  few  cases  some  phenomena  indicating  an  immunity, 
and  consequent  increased  phagocytic  power  of  the  leucocytes, 
from  an  immune  or  infected  person,  as  compared  with  the  normal, 
have  been  noticed.  This,  of  course,  is  quite  in  accordance  with 
MetchnikofPs  theoretical  views.  The  examples  are  not  numerous, 


PHAGOCYTOSIS  2QI 

and  the  best  is,  perhaps,  that  given  by  Bassett-Smith,  who  found 
that  in  Malta  fever  the  patient's  leucocytes  may  be  decidedly 
more  potent  than  normal  ones,  when  used  in  conjunction  with 
the  patient's  serum,  though,  when  normal  serum  is  used,  the 

difference  may  disappear.     Thus  : 

Cocci  per  Leucocytes. 

Patient's  serum  -f  patient's  leucocytes  +  emulsion  of  cocci  23-0  46-0 

,,              ,,      +  normal  leucocytes    -f          ,,                ,,  8'6  25-0 

Normal  serum    +  patient's  leucocytes  +          ,,                ,,  i6'o  30^0 

+  normal  leucocytes     +          ,,                ,,  19*0  29-0 

Rosenau  has  also  brought  forward  evidence  to  show  that 
leucocytes  from  cases  of  pneumonia  have  greater  phagocytic 
powers  than  those  from  healthy  persons,  and  are  less  easily 
killed  by  heat. 

Much  attention  was  attracted  of  late  by  Bail's  theory  of  the 
aggyessins.  Bail  found  that  if  washed  tubercle  bacilli  were  injected 
in  large  amount  into  the  peritoneum  of  guinea-pigs  infected  with 
tubercle,  the  animals  died  rapidly — i.e.,  in  eight  hours  or  so.1 
There  was  a  fluid  exudate  (containing  lymphocytes)  in  the  peri- 
toneal cavity,  and  this  exudate  (centrifugalized  to  get  rid  of  cells 
and  bacteria)  was  found  to  have  a  remarkable  action  in  increasing 
the  virulence  of  young  tubercle  bacilli  to  normal  animals.  Thus,  if 
a  few  cubic  centimetres  were  injected  together  with  the  bacilli, 
death  occurred  in  about  twenty  hours,  instead  of  in  some  weeks. 
He  found  that  this  virulence  was  apparently  due  to  an  inhibitory 
effect  which  the  fluid  exerted  on  phagocytosis.  When  bacilli 
were  injected  into  a  normal  animal  without  the  exudate,  many 
polynuclears  appeared  in  the  peritoneal  fluid  and  many  large 
mononuclear  cells,  and  many  of  the  bacilli  were  taken  up ;  but 
when  bacilli  and  exudate  were  injected,  few  cells  other  than 
lymphocytes  were  seen,  and  there  was  no  phagocytosis. 

These  observations  were  confirmed  and  extended  by  Bail  and 
others,  and  similar  phenomena  were  found  to  occur  in  the  case  of 
numerous  other  organisms,  if  not  in  all.  A  very  striking  example 
was  given  by  Weil  in  the  case  of  the  bacillus  of  chicken  cholera, 
which  is  extremely  virulent  to  rabbits,  so  that  a  millionth  of  a 
culture  (containing  perhaps  but  one  bacillus)  is  certainly  fatal. 
A  minute  trace  was  injected  into  the  pleura,  and  the  animal  died 
in  a  few  hours.  Several  cubic  centimetres  of  turbid  exudate,  the 

1  This,  of  course,  is  equivalent  to  the  tuberculin  reaction  in  an  extreme 
form. 

19—2 


2Q2  THE    AGGRESSINS 

cells  of  which  had  not  taken  up  any  bacteria,  were  collected,  and 
were  found  to  have  a  most  potent  effect  in  increasing  the  lethal 
action  of  the  organism.  This  could  not  be  tested  on  rabbits, 
since  they  were  loo  susceptible,  but  in  guinea-pigs  it  was  found 
to  lower  considerably  the  lethal  dose.  A  most  interesting  obser- 
vation was  made :  A  guinea-pig  which  had  received  a  small  dose 
of  a  culture  of  chicken  cholera,  and  had  apparently  recovered 
completely,  was  injected  eight  days  after  with  some  of  the  exudate, 
and  died  of  chicken  cholera  septicaemia,  showing  that  the  bacteria 
were  but  latent,  and  had  been  allowed  to  become  virulent  and 
active  in  virtue  of  the  action  of  the  exudate.  Further,  this  fluid, 
when  injected  into  rabbits,  was  found  to  immunize  them  against 
subsequent  injections  of  the  organism,  even  if  mixed  with  the 
exudate,  and  so  rendered  more  virulent. 

To  these  substances  Bail  gave  the  name  of  aggressins,  and 
considered  them  to  be  an  entirely  new  type  of  specific  substances 
formed  by  the  organism,  and  having  the  power  of  raising  its 
apparent  virulence  by  checking  phagocytosis  and  allowing  the 
invading  microbe  to  flourish  without  hindrance :  thus,  by  means 
of  the  concurrent  presence  of  its  specific  aggressin,  an  almost  in- 
nocuous organism,  such  as  B.  subtilis,  becomes  extremely  virulent. 
According  to  Bail  and  his  followers,  aggressins  are  only  formed  in 
vivo ;  but  this  is  denied  by  others,  who  claim  that  a  watery  emulsion 
of  certain  bacteria  has  many  at  least  of  their  peculiar  characters. 

Immunity  due  to  the  injection  of  aggressin  is  supposed  to  depend 
on  the  formation  of  a  specific  antibody,  or  anti-aggressin.  It  is 
produced  very  rapidly  after  the  injection  of  the  aggressin,  and  lasts 
several  weeks  or  more,  and  is  supposed  to  be  due  to  the  immediate 
neutralization  of  any  aggressin  which  the  bacterium  may  form  in 
vivo  by  the  anti-aggressin,  so  that  phagocytosis  in  unchecked. 

Agressins  are  sharply  specific,  except  perhaps  in  the  case  of 
those  for  B.  typhosis  and  B.  coli — i.e.,  the  injection  of  one  aggressin 
will  not  prevent  the  phagocytosis  of  any  species  of  bacterium 
other  than  that  by  the  action  of  which  it  was  prepared  ;  hence 
they  are  not  mere  leucocyte  poisons :  they  are  thermolabile. 

A  substance  which  prevents  phagocytosis  may  act  on  the  leuco- 
cyte, the  bacterium,  or  on  the  serum.  The  fact  just  described 
(that  phagocytosis  of  a  bacterium  A  can  go  on  in  the  presence  of 
an  aggressin  B)  shows  that  the  action  of  the  aggressins  is  not  on 
the  leucocytes.  Further,  as  Weil  and  Nikayama  have  shown, 
bacteria  which  have  been  acted  on  by  their  aggressins  and  the 


PHAGOCYTOSIS  2Q3 

latter  removed  by  washing,  are  readily  ingested.  The  action, 
therefore,  must  be  on  the  serum — i.e.,  aggressin  must  act  as  an 
anti-opsonin. 

This  leads  us  to  Wassermann  and  Citron's  explanation  of  the 
phenomena.  They  suppose  aggressins  to  be  simply  solutions  of  the 
bacterial  protoplasm  which  have  the  power  of  combining  with  the 
specific  protective  substances  of  the  animal,  and  so  disarming  its 
methods  of  defence.  In  other  words,  they  are  solutions  of  endo- 
toxin  of  feeble  toxicity.  This  view  is  strongly  supported — indeed, 
practically  proved — by  the  researches  of  Doerr,  who  found  that 
aggressins  caused  a  precipitate  when  mixed  with  their  specific 
immune  sera,  and  that  their  presence  might  bring  about  an 
absorption  of  the  complements,  just  as  if  they  were  free  bacterial 
receptors.  There  are  a  few  minor  differences  between  aggressins 
prepared  in  vivo  and  those  obtained  from  cultures  in  vitro,  but 
not  more  than  we  might  expect  from  the  differences  in  their  mode 
of  production. 

If  aggressins  are  merely  free  molecules  of  bacterial  protoplasm, 
we  should  expect  them  to  combine  with  opsonins,  just  as  do  the 
bacteria  themselves,  and  hence  to  act  as  anti-opsonins.  And  this 
supplies  a  striking  proof  of  the  specificity  of  the  opsonins,  for,  as 
already  stated,  an  aggressin  of  one  organism  (e.g.,  B.  coli)  does 
not  prevent  the  phagocytosis  of  another  organism  (e.g.,  B.  subtilis). 
This  must  apply  to  the  thermolabile  opsonin,  or  opsonin  proper, 
since  these  experiments  were  made  on  normal  animals. 

The  relationship  between  virulence  and  phagocytosis  is  an 
interesting  one.  As  a  general  rule,  it  will  be  found,  as  shown  by 
the  extensive  researches  of  Metchnikoff  and  his  school,  that  there 
is  an  inverse  ratio  between  the  two :  when  an  organism  is  viru- 
lent for  an  animal  it  will  be  ingested  by  the  leucocytes  to  a  very 
slight  extent,  and  vice  versa.  This  refers,  of  course,  mainly  to 
natural  immunity,  since  in  acquired  immunity  other  factors,  such 
as  the  action  of  bacteriolysins  or  antitoxins,  may  come  in.  There 
are,  however,  some  exceptions.  Thus,  tubercle  bacilli  injected 
into  the  peritoneum  of  normal  guinea-pigs  are  readily  taken  up  by 
the  phagocytes.  We  must  assume  in  this  case  that  an  organism 
may  be  taken  up  whilst  it  is  alive  and  uninjured,  that  it  may  be 
entirely  indigestible  by  the  leucocyte,  and  may  continue  to  grow 
and  multiply  in  its  interior.  This  is  also  sometimes  seen  in  acute 
infections :  the  common  localization  of  the  meningococcus  in  the 
polynuclear  leucocytes  is  well  known,  and  Andrewes  has  described 


2Q4  EFFECT    OF   VIRULENCE    ON    PHAGOCYTOSIS 

a  case  of  general  haemic  infection  by  this  organism  which  ran  a 
rapid  fatal  course  in  spite  of  all  the  organisms  (as  far  as  could  be 
seen)  being  taken  up  by  the  leucocytes.  In  general,  however,  the 
law  holds  good,  and  where  there  is  abundant  leucocytosis  the 
disease  tends  to  recovery  ;  when  there  is  little  or  none,  to  death. 

As  far  as  we  know  at  present,  the  failure  of  phagocytosis  which 
occurs  with  virulent  bacteria  is  due  to  their  deficient  opsonization  ; 
but  whether  this  is  because  they  require  a  large  dose  of  opsonin 
before  they  can  be  ingested,  or  whether  the  opsonin  cannot  com- 
bine with  them,  has  not  yet  been  determined  quite  satisfactorily. 
It  is  this  resistance  which  very  virulent  bacteria  exert  to  phago- 
cytosis which  causes  the  very  high  indices  seen  in  meningococcic 
infections.  If  the  index  is  determined  using  the  very  virulent 
organisms  recently  isolated  from  a  case  of  cerebro-spinal  fever, 
very  little,  if  any,  opsonization  and  phagocytosis  take  place  in 
the  specimen  in  which  normal  serum  is  used,  whereas  a  fair 
number  are  taken  up  when  the  serum  from  a  patient  is  employed. 
If,  however,  the  index  be  determined  using  an  old  laboratory 
culture,  much  more  phagocytosis  will  be  caused  by  normal  serum, 
and  the  index  will  be  nearer  unity.  The  relation  between  viru- 
lence and  lack  of  phagocytosis  is  discussed  subsequently  in  the 
section  on  immunity  to  bacteria. 

Lastly,  many  bacteria  form  toxins,  of  one  sort  or  another,  which 
prevent  phagocytosis  by  a  direct  action  on  the  leucocytes.  It  has 
been  shown  that  tetanus  spores  and  bacilli,  when  washed  per- 
fectly free  of  toxin,  are  quite  innocuous  to  all  animals,  and  are 
readily  taken  up  by  the  phagocytes  ;  the  presence  of  toxin,  it  may 
be  in  small  amounts,  by  killing  or  injuring  the  leucocytes,  allows 
the  bacilli  to  grow  in  the  tissues  and  elaborate  more  toxin.  Similar 
facts  probably  occur  in  the  case  of  diphtheria.  We  have  already 
referred  to  the  production  of  leucocidin  by  streptococci,  and  it  is 
obvious  that  when  this  is  formed  in  the  tissues  in  large  amount 
phagocytosis  will  be  reduced  or  stopped  altogether. 

The  nature  of  phagocytosis  requires  some  discussion.  We  are, 
perhaps,  rather  too  apt  to  be  influenced  by  the  readily  observed 
phenomena  of  ingestion  of  bacteria,  diatoms,  etc.,  by  amoebae, 
and  to  assume  that  it  is  in  all  cases  an  active  process  on  the  part 
of  the  leucocytes,  which  are  usually  considered  to  approach  their 
prey  by  active  movements  directed  by  positive  chemotaxis,  and 
to  seize  them  by  means  of  their  pseudopodia.  Chemotaxis  does, 
of  course,  occur  in  the  tissues,  but  it  is  clear  that  it  does  not  take 


PHAGOCYTOSIS  2Q5 

place  in  the  artificial  conditions  of  opsonin  estimations,  where  the 
bacteria  are  uniformly  distributed  throughout  the  fluid,  and  there 
is  no  reason  why  the  leucocyte  should  be  attracted  in  one  direc- 
tion rather  than  in  another  ;  and  movement  of  leucocytes  either 
does  not  occur  at  all  or  does  so  only  to  a  very  minute  extent  in 
saline  solution.     It  takes  place  much  more  actively  in  unheated 
serum — a  fact  which  gives  some  support  to  the  theory  of  stimulins, 
previously  mentioned,  but  not  discussed.    It  is  quite  possible  that 
all  the  facts  related  concerning  opsonic  action  may  be  due  to  one 
or  more  substances  which  occur  in  the  serum,  and  which  have 
the  power  of  stimulating   the  leucocyte,   or  of  altering  it  in  a 
manner  to  be  discussed  subsequently.     The  phenomena  of  the 
phagocytosis   of   sensitized    bacteria   in   normal   saline   solution 
would,  of  course,  be  due  to  a  liberation  of  this  stimulin  from  its 
combination  with   the   bacteria.     This   is   known    to   occur,  for 
sensitized  bacteria  will  yield  some  opsonin  on  prolonged  soaking 
in  normal   saline  or  heated   serum ;    the  fluid   acquires  opsonic 
properties,  and  the  bacteria  becomes  insensitive  to  phagocytosis. 
As  Sellards  points  out,  the  fact  that  unorganized  bodies,  such  as 
carmine,  particles  of  carbon,   melanin,  etc.,  are  taken  up  more 
readily  in  the  presence  of  fresh  serum  is  somewhat  in  favour  of 
this  view.    It  is  difficult  to  think  that  these  substances  are  affected 
in  a  way  similar  to  bacteria  or  other  antigens  when  combined 
with  their  specific  antibodies.     There  appears  to  be  no  crucial 
test  for  determining  the  point. 

And  there  is  some  reason  for  thinking  that  the  actual  process 
of  phagocytosis  may  be  a  physical  one,  akin  to  agglutination,  and 
entirely  independent  of  any  movements  or  other  vital  processes 
on  the  part  of  the  leucocytes.  The  chief  evidence  in  favour  of 
this  view  arises  from  the  fact  that  phagocytosis  may  occur  under 
conditions  in  which  no  movements  of  any  sort  take  place.  This 
was  first  pointed  out  by  Ledingham  in  a  series  of  important 
researches  on  the  relation  between  temperature  and  opsonization. 
He  showed  that  when  a  series  of  opsonin  mixtures  were  incu- 
bated at  temperatures  varying  between  18°  and  37°  C.,  the 
latter  temperature  brought  about  much  more  phagocytosis  than 
the  former ;  and,  further,  that  at  the  latter  point  there  was  very 
little  difference  in  the  index  between  preparations  incubated  for 
fifteen  or  thirty  minutes,  while  in  the  former  there  was  a  long  latent 
period  in  which  but  little  phagocytosis  occurred.  This  he  showed 
to  be  due  to  the  fact  that  opsonin  combines  with  bacteria  but  very 


2Q6  TEMPERATURE    AND    OPSONIZATION 

slowly  at  1 8°  C.  and  rapidly  at  37°  C.  Provided  the  bacteria 
were  sensitized  at  the  latter,  it  mattered  little  or  nothing  whether 
the  mixture  were  incubated  at  either  temperature,  and  a  very 
considerable  amount  of  phagocytosis  took  place  as  low  as  10°  C. 
Now  at  this  point  no  movements  of  any  sort  occur,  and  it  is  quite 
easy  to  satisfy  oneself  by  actual  observation  under  the  microscope 
that  bacteria  opsonized  at  37°  C.  may  be  taken  up  at  a  low 
temperature  by  Bacteria  which  remain  absolutely  motionless 
during  the  process.  This  is  even  more  easily  observed  by  using 
a  modification  of  a  method  recently  introduced  by  Ponder,  and 
of  very  great  value  in  the  direct  observation  of  phagocytic  and 
other  phenomena.  If  a  drop  of  blood  be  placed  in  a  glass  cell 
about  o'2  millimetre  deep  (such  as  is  used  in  mounting  diatoms, 
etc.,  in  fluid),  covered  with  a  cover-glass,  and  incubated  for  fifteen 
minutes  or  so,  both  slide  and  cover-glass  will  be  found  to  be 
dotted  about  with  leucocytes  which  adhere  so  firmly  that  all  the 
red  corpuscles  can  be  washed  off  with  warm  normal  saline  solu- 
tions, leaving  the  leucocytes  adherent  to  the  glass.  If  now  the 
cell  be  filled  with  serum  mixed  with  bacteria,  and  incubated  at 
37°  C.,  or  with  bacteria  thus  opsonized  and  thoroughly  washed, 
the  process  of  phagocytosis  can  be  readily  watched,  and  is  seen 
to  take  place  at  18°  C.  or  lower.  Under  these  circumstances,  no 
active  movement  or  protrusion  of  pseudopodia  takes  place  at  all, 
and  it  is  easy  to  watch  a  sensitized  coccus  being  gradually 
attracted  to  and  absorbed  into  the  body  of  the  leucocytes.  The 
process  strongly  recalls  the  agglutination  of  bacteria.  A  coccus 
lying  within  a  certain  distance  of  the  cell  is  seen,  like  the  others, 
to  be  in  active  Brownian  movement,  and  the  appearances  would 
suggest  that  it  is  slightly  more  easy  for  it  to  move  towards  the 
cell  than  away  from  it;  It  oscillates  in  all  directions,  but 
gradually  approaches  nearer  and  nearer  the  leucocyte,  and  is 
finally  taken  in.  Similar  phenomena  can  be  seen  (using  a  hot 
stage)  when  sensitized  bacteria  in  an  emulsion  in  normal  saline 
solution  are  added  to  leucocyte  films  at  37°  C. ;  and  here  also 
no  movement,  or  but  little,  takes  place.  If,  however,  serum  be 
added,  there  is  usually  some  movement  of  the  pseudopodia,  but 
little  or  no  locomotion  from  place  to  place. 

It  is  not  easy  to  determine  whether  phagocytosis  may  take 
place  in  dead  leucocytes.  I  have  not  been  able  to  detect  it  in 
leucocytes  killed  either  by  heat  or  cold,  but  Rosenau  states  that 
when  leucocytes  killed  in  the  former  way  are  mixed  with  opsonized 


PHAGOCYTOSIS  2Q7 

cocci,  they  collect  round  the  cell,  though  they  are  not  actually 
ingested,  and  this  is  confirmed  by  Sellards.  Killed  leucocytes 
probably  undergo  a  sort  of  coagulation  equivalent  to  rigor  mortis, 
which  would  prevent  the  ingress  of  bacteria. 

Sellards  has  shown  that  salts  are  as  necessary  for  phagocytosis 
as  for  agglutination.  The  isotonic  solution  in  which  the 
leucocytes  were  suspended  was  5-5  per  cent,  of  saccharose ;  the 
bacteria  were  opsonized  by  fresh  serum,  washed  thoroughly,  and 
suspended  in  the  same  sugar  solution.  Little  or  no  phagocytosis 
occurred,  but  it  took  place  if  salts  were  added.  This,  again,  does 
not  look  like  a  vital  process,  but  is  quite  analogous  with  agglutina- 
tion, in  which  we  have  every  reason  to  believe  that  the  effect  is 
due  to  an  alteration  of  surface  tension.  So  also  with  the  action  of 
serum  in  aiding  the  phagocytosis  of  substances  such  as  carmine  or 
carbon.  We  have  only  to  suppose  that  some  substance  is  occluded 
on  the  surface  of  the  inert  substance,  the  surface  tension  of  which  it 
alters  in  the  same  way  as  opsonin  alters  that  of  the  bacteria. 

The  degree  of  opsonization  is  determined  to  some  extent  by  the 
amount  of  salt  present,  and  is  found  to  be  least  (in  the  absence  of 
serum)  in  a  1*2  per  cent,  solution  ;  hence  this  strength  of  salt  is 
used  by  some  observers  in  opsonic  determinations  in  order  to 
reduce  the  amount  of  spontaneous  phagocytosis  as  low  as  possible. 
Hamburger  and  Hekma  have  also  shown  that  a  minute  trace  of 
calcium  chloride  has  a  great  influence  in  increasing  the  opsonic 
power  of  the  serum  (we  have  already  seen  that  it  aids  the  ag- 
glutination of  cholera  vibrios),  and  that  the  activity  of  the  serum 
is  increased  by  alkalis  and  diminished  by  acids.  Chloride  of 
potassium,  unlike  chloride  of  sodium,  has  also  an  unfavourable 
effect  on  the  leucocytes. 

If  we  push  our  investigations  a  little  farther,  we  may  perhaps  be 
led  to  the  belief  that  the  amoeboid  movements  and  protrusion  of 
pseudopodia  which  leucocytes  display  under  suitable  circumstances 
may  themselves  be  effects  of  surface  tension  rather  than  strictly 
vital  phenomena.  Consider  the  case  of  the  film  of  blood  prepared 
by  Ponder's  method,  or  by  the  use  of  a  glass  cell,  as  recommended 
above.  When  this  is  incubated,  large  numbers  of  leucocytes 
appear  both  on  the  lower  and  upper  surfaces.  Now  in  the  latter 
case  the  effect  cannot  be  due  to  gravity,  for  the  leucocytes  are 
heavier  than  the  serum.  It  would  be  too  great  a  strain  on  the 
imagination  to  suppose  the  leucocytes  capable  of  actual  swimming 
movements  through  the  blood  (and  it  may  be  remarked  that  many 


2g8  PHYSICAL    EXPLANATION    OF    PHAGOCYTOSIS 

find  their  way  to  the  top  before  coagulation  occurs,  though  the 
process  appears  to  continue  after  that),  and  the  only  alternative  is 
a  physical  attraction  between  the  glass  and  the  leucocytes.  This 
is  a  perfectly  feasible  explanation,  and  if  it  is  true  the  next  stage 
in  the  process  would  necessarily  follow.  This  is  the  flattening 
out  of  the  leucocytes,  so  that  they  form  thin  plaques  of  very  much 
larger  diameter  than  the  same  cells  as  seen  in  ordinary  wet  films. 
This  is  very  difficult  to  explain  as  any  vital  effect,  but  it  is 
exactly  what  we  should  expect  to  happen  if  the  leucocyte  (which, 
like  all,  or  almost  all,  forms  of  living  protoplasm,  is  to  be  regarded 
as  a  liquid)  were  pulled  out  under  the  influence  of  surface  tension, 
just  as  a  drop  of  liquid  paraffin  is  stretched  out  into  an  infinites!  - 
mally  thin  film  when  dropped  on  the  surface  of  water. 

The  bizarre  forms  which  the  leucocytes  assume  in  a  preparation 
made  by  Ponder's  method,  with  long  pseudopodia,  are  explicable 
on  the  assumption  that,  owing  to  irregularities  in  the  cover-glass, 
the  surface  tension  is  not  uniform  in  all  directions,  or  'that  the 
protoplasm  of  the  leucocyte  is  not  of  the  same  degree  of  viscidity 
throughout.  Similar  irregular  protuberances  can  be  produced  in 
globules  of  oil  or  water  by  purely  physical  means,  and  Pauli  goes 
so  far  as  to  say  that  "  since  the  discovery  of  the  amoeboid  move- 
ments of  oil  droplets,  and  the  careful  physical  analysis  of  this 
process  by  Quincke,  the  formation  of  pseudopodia  has  been 
robbed  of  the  characteristics  of  a  specific  life  phenomenon,  and 
later  investigations  have  shown  that  it  is  governed  in  all  its 
details  by  the  laws  of  surface  tension.  The  taking  up  of  food  and 
the  process  of  defalcation  in  rhizopods  can  also  be  explained  in 
the  same  way."  The  process  of  the  ingestion  of  an  opsonized 
bacterium  suspended  in  serum  at  the  body  temperature,  in  which 
it  is  occasionally  possible  to  see  the  protrusion  and  seizure  of  the 
organism  by  a  long,  slender,  and  flexible  pseudopodium,  is 
explicable  as  follows  :  Owing  to  the  change  of  surface  tension 
induced  by  the  action  of  the  opsonin  on  the  bacterium,  there 
is  generated  an  attractive  force  which  tends  to  draw  the  two 
together.  The  leucocyte,  being  fixed  to  the  cover-glass  like  a 
sucker,  does  not  move,  but  a  small  portion  of  its  substance,  being 
liquid  or  semi-liquid  in  consistency,  is  drawn  out  until  it  meets 
the  bacterium,  which  is,  of  course,  also  attracted.  The  two  meet, 
and  then  it  will  be  found  that  the  organism  is  firmly  held  in 
contact  with  the  pseudopodium,  so  that  it  is  not  released  even  if 
the  latter  be  carried  to  and  fro  by  currents  in  the  fluid. 


PHAGOCYTOSIS  2QQ 

The  effects  of  surface  tension  may  also  be  traced  in  some  of 
the  phenomena  of  inflammation,  especially  in  the  adhesion  of  the 
leucocytes  to  the  vessel  wall.  It  has  been  abundantly  shown 
that  this  is  due  to  an  alteration  in  the  latter,  and  it  appears  likely 
that  this  is  simply  due  to  a  change  in  the  tension  developed  at 
the  surface  between  the  endothelial  lining  and  the  serum,  in 
virtue  of  which  the  former  behaves  like  the  glass  in  Ponder's 
method,  attracting  the  leucocyte  and  causing  it  to  adhere  and 
flatten  itself  out.  This  extension,  so  as  to  offer  as  large  a  surface 
as  possible,  which  is  displayed  by  the  leucocytes,  and  especially 
of  the  polynuclears,  when  they  come  into  contact  with  a  resistant 
surface,  was  noted  long  ago  by  Massart  and  Bordet,  and  in  virtue 
of  it  they  are  able  to  make  their  way  through  the  finest  pores, 
even  in  compact  bodies  like  bone  and  ivory.  The  remarkable 
deformation  in  shape  which  leucocytes  undergo  in  acutely  inflamed 
tissues  is  not  usually  appreciated.  It  was  pointed  out  to  me  by 
Whitfield,  and  may  often  be  seen  at  the  edge  of  the  sections 
where  the  fixation  is  perfect,  provided  the  material  has  been 
placed  in  the  fixing  fluid  immediately  after  its  excision.  The 
polynuclear  leucocytes  are  often  overlooked  altogether,  being 
pulled  out  into  long  strands  of  protoplasm  containing  nuclear 
filaments,  giving  the  section  a  remarkable  mossy  appearance. 
This  change  in  the  surface  tension  of  the  vessels,  lymph  clefts, 
etc.,  probably  plays  a  part  of  great  importance  in  diapedesis.  It 
is  somewhat  doubtful,  however,  whether  it  can  afford  a  complete 
explanation  of  the  phenomena  of  chemotaxis,  in  which  a  vital 
and  apparently  quasi-intelligent  action  appears  probable. 

It  must  not  be  imagined  that  the  vitality  of  the  leucocyte  is  to 
be  regarded  as  unimportant  in  the  consideration  of  phagocytosis 
as  a  means  of  defence.  Here  the  process  has  only  begun  when 
the  organism  is  ingested,  and  unless  suitable  digestive  ferments 
are  secreted,  the  bacterium  dissolved,  and  the  endotoxin  absorbed 
or  otherwise  dealt  with,  the  process  is  useless,  or,  by  carrying 
bacteria  out  of  the  lesion  to  other  parts  of  the  body,  may  even  be 
harmful. 


CHAPTER  XI 
"REACTIONS"  AND  SIMILAR  PHENOMENA 

NOT  long  after  the  discovery  of  the  tubercle  bacillus  Koch  found 
that  the  effects  of  an  inoculation  of  living  cultures  of  the  organism 
were  quite  different  in  normal  and  in  tuberculous  animals.  If  a 
normal  animal  is  inoculated  by  scarification  of  the  skin  the 
wound  soon  heals,  and  in  about  a  fortnight  a  hard  nodule  forms. 
This  ulcerates,  and  remains  an  open  ulcer  until  the  animal  dies. 
If  a  second  inoculation  be  made  after  the  first  has  run  its  course 
to  the  stage  of  ulceration,  the  process  is  profoundly  modified.  No 
nodule  is  formed  at  the  site  of  the  second  inoculation,  but  the 
tissue  round  the  first  becomes  hard,  dark-coloured,  and  finally 
necrotic,  and  may  be  shed  en  masse  and  the  lesion  undergo  com- 
plete cure.  Koch  found,  further,  that  this  change  might  be 
brought  about  by  injections  of  dead  cultures  even  after  they  had 
been  boiled.  He  found,  too,  that  a  large  dose  of  these  killed 
cultures  (which  would  cause  nothing  but  local  suppuration  in 
normal  animals)  would  kill  a  tuberculous  guinea-pig  in  a  short 
time — six  to  forty-eight  hours — the  symptoms  being  fever,  acute 
inflammation,  running  on  to  necrosis,  in  the  region  of  the  tubercu- 
lous lesions,  and  in  some  cases  generalization  of  the  bacilli 
throughout  the  body.  When  very  minute  doses  were  used  he 
found,  on  the  contrary,  that  improvement  might  occur,  and  the 
tuberculous  ulcer  become  cicatrized  over. 

This  was  made  the  basis  of  a  method  for  the  treatment  of 
tubercle  in  man.  But  Koch  found  the  use  of  killed  cultures 
inconvenient,  since  the  bacilli  wrere  but  slowly  absorbed,  and 
might  give  rise  to  abscesses.  He  argued  that  the  effect  was 
evidently  due  to  some  soluble  substance  which  diffused  out  of  the 
bacilli,  and  after  long  research  prepared  the  substance  which  is 
now  so  familiar  as  the  old  tuberculin.  It  is  a  solution  in  40  to  50  per 
cent,  glycerin  of  the  soluble  products  of  the  tubercle  bacillus,  and 
is  prepared  by  cultivating  that  organism  for  several  weeks  in 

300 


AND    SIMILAR   PHENOMENA  3OI 

glycerinated  veal  broth  in  a  thin  layer,  so  that  there  is  an  abundant 
supply  of  oxygen.  This  culture  is  evaporated  to  one-tenth  of  its 
volume  and  filtered  through  a  Chamberland  filter.  There  are 
numerous  slight  modifications  in  the  process  of  manufacture, 
but  they  are  unimportant. 

Old  tuberculin  is  a  syrupy  brownish-yellow  fluid,  with  a  faint 
aromatic  smell.  It  contains  peptones  and  traces  of  other  proteid 
bodies,  but  the  nature  of  the  substance  on  which  its  extraordinary 
power  depends  is  quite  unknown.  It  is  in  a  sense  to  be  regarded 
as  a  toxin  of  the  tubercle  bacillus,  but  it  is  not  a  true  toxin,  like 
those  of  diphtheria  and  tetanus,  since  it  is  practically  non-toxic  for 
healthy  animals  or  for  man.  Its  injection  in  large  quantity  may 
cause  a  slight  febrile  reaction,  but  not  much  more  than  a  similar 
injection  of  peptones,  etc.,  from  any  other  source.  It  differs,  also, 
in  a  marked  degree  from  the  exotoxins  in  that  it  is  not  destroyed 
by  a  temperature  of  100°,  or  even  of  120°  C.  It  is  dialyzable. 

When  injected  into  tuberculous  animals  it  causes  the  same 
"  reaction  "  as  was  produced  by  the  living  or  dead  culture,  and  this 
in  very  minute  amount.  A  dose  of  i  milligramme  will  cause  a  sharp 
reaction  in  a  tuberculous  patient,  and,  indeed,  one-tenth  of  that 
amount  will  sometimes  suffice.  When  we  consider  that  the 
material  consists  mainly  of  the  nutrient  ingredients  of  the  broth — 
Koch  thought  that  the  active  principle  might  form  i  per  cent,  of  the 
whole — its  extraordinary  potency  is  evident. 

The  phenomena  of  the  "  reaction  "  are  as  follows  :  There  may 
be,  but  usually  is  not,  some  inflammatory  oedema  at  the  seat  of 
injection.  The  temperature  rises  precipitously,  often  reaching 
105°  F.  in  a  few  hours,  and  falls  almost  as  quickly.  With  this 
there  are  the  usual  symptoms  of  fever,  malaise,  shivering,  etc. 
This  is  the  general  reaction.  The  local  reaction  occurs  round  the 
pre-existing  tuberculous  lesion,  and  is  best  seen  in  lupus,  tubercu- 
lous ulcers,  etc.  Its  severity  depends  upon  the  dose  given.  With 
a  small  dose  there  is  a  little  redness  and  swelling  and  some  mild 
inflammatory  oedema,  the  whole  lasting  but  a  day  or  two.  W^hen 
it  subsides  the  lesion  often  undergoes  great  improvement.  After 
larger  doses  the  local  reaction  is  more  marked,  acute  inflammation 
occurs,  the  tissues  in  and  around  the  tuberculous  foci  undergo 
coagulation  necrosis,  and  are  cast  off.  When  this  takes  place  in 
the  skin  it  may  lead  to  complete  cure,  but  in  the  internal  organs 
it  is  a  source  of  grave  danger,  often  leading  to  dissemination  of  the 
bacilli  and  a  consequent  general  infection.  This  occurred  in  the 


302 


THE    TUBERCULIN    REACTION 


early  days  of  the  use  of  the  fluid,  when  it  was  hailed  as  a  specific 
cure  for  the  disease,  and  Koch's  limitations  of  its  use  ignored.  At 
present  it  is  used  as  a  method  of  diagnosis,  and  found  to  be  of 
great  value  and  devoid  of  danger  if  used  with  proper  precautions. 
And  there  can  be  no  doubt  that  the  bad  results  obtained  when  the 


Normal 
98° 


FIG.  70. — SEVERE  TUBERCULIN  REACTION  IN  A  CASE  OF  BAZIN'S  DISEASE. 
(Under  Dr.  Whitfield.) 

potentialities  of  the  substance  were  so  little  known  have  led  to  its 
being  unjustly  abandoned  as  a  method  of  cure.  Properly  applied 
to  suitable  cases,  it  has  proved  of  great  value. 

The  reaction  is  a  specific  one,  except  that  it  is  sometimes  given 
in  patients  with  syphilis,  leprosy,  or  actinomycosis.  This  is 
unusual. 

When  patients  are  treated  with  gradually  increasing  doses  of 
tuberculin  they  become  partially  immunized,  so  that  no  febrile 


"  REACTIONS        AND    SIMILAR    PHENOMENA  303 

reaction  is  caused  by  large  doses.  Thus  Wassermann  records  a 
case  in  which  300  milligrammes  caused  no  reaction.  The  patient 
had  been  treated  for  a  year,  the  dose  being  gradually  increased. 
It  appears,  too,  that  by  careful  treatment  of  animals  an  antituber- 
culin  can  be  produced  which  has  the  power  of  inhibiting  the  effects 
of  tuberculin  in  tuberculous  animals.  It  has,  however,  little  or 
no  effect  in  the  treatment  of  the  disease — another  proof  that  tuber- 
culin is  not  the  specific  toxin. 

Quite  recently  important  modifications  have  been  introduced  in 
the  diagnostic  application  of  tuberculin.  Von  Pirquet's  reaction, 
or  the  cnti-veaction,  is  elicited  by  placing  a  drop  of  tuberculin 
(undiluted  or  a  25  per  cent,  solution)  on  the  skin,  and  performing 
scarification  just  as  for  an  ordinary  Jennerian  vaccination.  It  is 
advisable  to  make  a  similar  control  scarification  without  using 
tuberculin  in  order  that  the  lesions  may  be  compared.  The 
simple  inoculation  shows  a  little  redness,  which  soon  disappears. 
That  made  with  tuberculin  does  the  same  if  the  patient  is  not 
tuberculous.  If  he  is,  a  small  red  papule  is  formed,  which  increases 
for  three  or  four  days  and  disappears  in  a  week  or  so. 

Calmette's  method,  the  ophthalmo  -  reaction,  is  obtained  by 
instilling  one  or  two  drops  of  diluted  tuberculin  into  the  conjunc- 
tival  sac.  He  recommends  the  use  of  old  tuberculin  which  has 
been  precipitated  with  absolute  alcohol  and  redissolved  in  distilled 
water,  as  being  less  irritating  and  less  likely  to  cause  a  pseudo- 
reaction  in  a  non-tuberculous  patient.  The  reaction  in  this  case 
consists  of  a  mild  attack  of  conjunctivitis,  lasting  twenty- four 
hours,  and  accompanied  by  redness  and  swelling  of  the  caruncle 
and  a  small  amount  of  mucoid  secretion.  The  reaction  should  be 
over  in  twenty-four  hours,  but  in  some  cases  undesirable  results 
have  occurred  from  a  secondary  infection  with  organisms  capable 
of  causing  a  more  severe  conjunctivitis  or  keratitis.  For  this 
reason  the  test  should  be  used  with  care,  if  at  all. 

These  tests  are  most  applicable  in  children,  since  in  adults  the 
frequency  of  cured  tubercle,  also  leading  to  hypersensitiveness, 
may  lead  to  reactions  where  there  is  no  clinical  tubercle,  or  the 
patients  may  be  more  or  less  immune. 

-  Reactions  are  given  in  other  diseases,  the  most  important  being 
glanders.  Mallein  is  a  fluid  obtained  from  cultures  of  B.  mallei 
exactly  as  tuberculin  is  obtained  from  tubercle  bacilli.  It  is  non- 
toxic  to  normal  animals,  but  it  causes  a  febrile  reaction  in  those 
infected  by  glanders,  even  if  there  is  but  a  small  latent  lesion. 


304  OTHER    REACTIONS 

There  is  also  a  local  reaction  at  the  site  of  the  lesion,  though  this 
is  less  marked  than  in  tubercle.  There  is,  however,  a  very 
marked  production  of  inflammatory  oedema  at  the  site  of  the 
inoculation,  and  this  furnishes  the  most  certain  test  for  the 
disease.  A  hard  raised  mass  is  formed,  which  increases  in  size 
for  twenty-four  hours  or  more,  becoming  as  large  as  the  palm  of 
the  hand,  and  persists  some  days.  It  often  gradually  travels 
down  the  neck  (in  the  horse),  as  if  under  the  action  of  gravity. 

It  will  be  noticed  that  an  essential  feature  in  these  reactions  is 
the  rapid  development  of  symptoms  after  an  inoculation  or 
injection  in  a  subject  already  infected  with  the  disease.  Von 
Pirquet  has  brought  forward  other  examples,  which,  if  less 
dramatic  in  their  course  than  Koch's  phenomenon,  are  at  least 
comparable  with  the  cuti-reaction.  Thus  it  has  been  noticed  that 
the  second  (Jennerian)  vaccination,  practised  some  years  after  the 
first,  runs  a  rapid  course.  Von  Pirquet  has  shown  that  if  a 
revaccination  be  made  a  few  months  after  the  first,  the  reaction 
takes  place  in  twenty-four  hours.  It  does  not,  of  course, 
develop,  in  the  same  way  as  a  primary  vaccination,  but  a  small 
papule,  often  surrounded  by  an  areola,  makes  its  appearance,  and 
lasts  some  two  or  three  days. 

Chantemesse  has  observed  in  typhoid  fever  phenomena  similar 
to  those  seen  in  Calmette's  ophthalmo-reaction  in  tubercle.  He 
instils  a  single  drop  of  typhoid  "toxin"  (obtained  by  cultivating 
typhoid  bacilli  in  extract  of  spleen  digested  with  pepsin),  and 
finds  that  in  normal  persons  there  is  but  a  little  transient  redness, 
whilst  in  typhoid  patients  there  is  redness,  lachrymation,  and  the 
formation  of  a  sero-fibrinous  exudate.  The  process  attains  its 
maximum  in  six  to  twelve  hours.  He  makes  use  of  this  reaction 
as  a  method  of  diagnosis. 

The  phenomena  of  the  "  negative  phase,"  seen  probably  in  all 
antibodies,  but  specially  studied  in  connection  with  the  opsonins, 
are  probably  similar  in  nature  to  these  reactions,  although  the 
doses  of  vaccines  given  are  usually  so  small  that  the  clinical 
manifestations  do  not  appear.  Sometimes,  however,  this  does 
happen.  Thus  Irons  found  that  a  dose  of  500,000,000  dead 
gonococci  caused  no  reaction  in  healthy  persons ;  but  if  given  to 
patients  already  suffering  from  a  gonococcal  infection,  it  produced 
fever,  pains  in  the  joints,  and  general  malaise.  In  most  cases  the 
difference  between  the  behaviour  of  a  healthy  and  infected  person 
or  •  animal  is  traceable  solely  in  the  variations  of  the  opsonic 


"  REACTIONS  "   AND    SIMILAR   PHENOMENA  305 

index.  When  an  ordinary  dose  of  any  vaccine  is  given  to  a 
healthy  person  the  opsonic  index  undergoes  but  slight  changes, 
and  in  particular  there  is  no  fall  or  negative  phase.  There  may 
be  a  slight  subsequent  rise.  When  the  same  dose  is  given  to  a 
person  infected  with  the  same  organism,  the  negative  phase 
(perhaps  preceded  by  a  "  false  rise ")  is  most  marked,  and  is 
followed  by  a  positive  rise,  or  sometimes  by  a  series  of  rises  and 
falls,  gradually  dying  away  like  a  wave.  Similar  phenomena  can 
be  produced  in  a  healthy  person,  but  here  the  dose  must  be  much 
larger.  Evidently,  therefore,  the  presence  of  an  infecting  agent 
other  than  tubercle  causes  a  condition  of  unstable  equilibrium,  in 
which  the  tissues  react  in  a  different  manner  to  healthy  ones. 
And  the  same  condition  of  altered  sensitiveness  may  persist  for 
long  after  the  disease  or  injection  of  a  vaccine,  so  that  a  dose  of 
dead  bacilli  that  has  but  little  action  in  health  causes  a  great 
output  of  antibodies.  This  reaction  appears  to  be  a  general  one, 
occurring  with  bacteriolysins,  agglutinins,  etc. 

Attempts  have  naturally  been  made  to  account  for  a  phe- 
nomenon so  remarkable  as  the  tuberculin  reaction,  and  the  large 
number  of  explanations  suggest  that  none  is  altogether  satisfactory. 
Many  of  them  do  not  call  for  notice. 

Koch's  explanation,  which  was  put  forward  more  as  a  working 
hypothesis  than  as  an  established  fact,  was  this :  The  bacillus 
formed  a  toxin,  which  diffused  outwards  from  the  colonies  in  the 
tissues,  and  when  in  a  sufficient  state  of  concentration  set  up  a 
coagulation  -  necrosis  going  on  to  caseation.  In  the  zone  of 
tissues  just  beyond  this  region  the  necrosis-producing  substance  is 
present,  but  not  in  a  sufficient  degree  of  concentration  to  kill  the 
tissues.  The  injection  of  a  little  more  of  this  substance— ?.£.,  of 
tuberculin — is  sufficient  to  turn  the  scale,  and  a  rapid  increase  of 
the  necrosis  takes  place.  He  explains  the  beneficial  effects  of 
the  treatment  in  this  wise :  The  necrotic  tissue  does  not  form  a 
suitable  medium  of  growth  for  the  tubercle  bacillus  (which  is  but 
rarely  seen  in  caseous  material),  and  the  extension  of  the  process 
may  lead  to  the  complete  enclosure  of  the  bacteria  in  dead  and 
altered  tissues,  in  which  they  are  incapable  of  further  growth. 

This  theory  assumes  that  the  substance  which  produces  necrosis 
is  identical  with  the  active  principle  of  tuberculin  ;  but  tuberculin 
in  large  doses  will  not  produce  necrosis  in  a  healthy  animal.  It 
seems  also  to  fail  to  account  for  the  remarkable  rise  in  the 
temperature,  since  it  occurs  in  patients  who  are  not  febrile,  as 

20 


306       EXPLANATIONS    OF   THE    TUBERCULIN    REACTION 

we  should  expect  them  to  be  if  tuberculin  were  diffusing  from 
their  lesions. 

Ehrlich's  views  are  quite  similar  to  Koch's,  and  he  regards  the 
reaction  as  due  to  the  effect  of  the  tuberculin  on  tissues  which  are 
injured  by  it  at  the  time  of  the  injection,  and  in  which  a  slight 
extra  dose  is  sufficient  to  turn  the  scale. 

Others  have  thought  that  the  reaction  is  indicative  of  a  hyper- 
sensitiveness  of  the  patient  to  tuberculin,  using  the  term  in  the 
sense  in  which  we  employed  it  in  dealing  with  the  toxins.  This, 
of  course,  is  true,  but  it  scarcely  seems  a  sufficient  explanation  in 
itself.  We  shall  revert  to  the  subject  after  giving  an  account  of 
some  most  remarkable  discoveries  that  have  recently  been  made 
concerning  this  subject. 

Marmorek  holds — in  all  probability  correctly — that  tuberculin 
is  not  to  be  regarded  as  in  any  sense  the  true  toxin  of  the  tubercle 
bacillus.  This  is  only  formed  when  the  organism  is  living  para- 
sitically  in  the  tissues,  or  in  artificial  conditions  bearing  a  very 
close  approximation  thereto,  not  in  such  a  simple  medium  as  plain 
broth.  Tuberculin  has  this  effect  on  a  tuberculous  animal :  it 
stimulates  the  tubercle  bacilli  to  a  sudden  and  energetic  production 
of  toxin,  which  gives  rise  to  the  local  reaction,  and,  passing  into 
the  vessels,  to  fever  and  its  concomitant  general  phenomena. 
There  is  nothing  inherently  improbable  in  this  suggestion,  except 
that  no  reason  is  forthcoming  as  to  the  way  in  which  tuberculin 
exerts  this  very  remarkable  action,  but  there  is  little  direct  evidence 
in  its  favour.  The  toxin  which  Marmorek  claims  to  have  pro- 
duced by  the  application  of  this  principle  is  so  weak  as  not  to 
be  worth  calling  a  toxin. 

Wassermann  and  Briick  point  out  that  the  extremely  minute 
amount  which  must  be  present  in  the  blood  at  a  given  time  leads 
to  the  supposition  that  the  tuberculin  injected  must  leave  the  blood- 
stream and  become  concentrated  in  the  region  of  the  tuberculous 
focus.  Thus,  if  a  person  with  5,000  c.c.  of  blood  reacts  to  an  injec- 
tion of  i  milligramme  of  tuberculin,  the  dilution  will  be  i  :  5,000,000, 
and  they  find  that  this  dilution  injected  directly  into  a  tuberculous 
lesion  gives  absolutely  no  reaction.  They  then  proceed  to  argue 
that  this  attraction  of  the  tuberculin  from  the  blood  must  be  due 
to  the  presence  in  the  tuberculous  tissue  of  an  antitoxin  or  anti- 
tuberculin.  They  investigated  the  presence  or  absence  of  this 
substance  by  means  of  the  method  of  fixation  of  the  complements. 
Extracts  of  tuberculous  tissues,  when  mixed  with  tuberculin, 


AND    SIMILAR   PHENOMENA  307 

acquired  the  power  of  absorbing  haemolytic  complements  from 
fresh  serum,  and  so  of  inhibiting  the  haemolysis  of  sensitized  red 
corpuscles.  Extracts  of  normal  organs  had  no  such  power. 

This  is  made  the  basis  for  their  theory  of  the  reaction.  The 
injected  tuberculin  circulates  in  the  blood  until  it  reaches  the 
antituberculin  present  in  the  lesions.  The  two  combine,  and  in 
doing  so  attract  the  complements  which  we  must  suppose  to  be 
free  in  the  plasma.  This  fixation  is  supposed  to  be  followed  by 
cytolysis  of  the  cells  of  the  part.  This  accounts  for  the  local 
reaction.  In  this  solution  of  the  tissue  cells  products  of  disintegra- 
tion are  set  free,  pass  into  the  blood,  and  give  rise  to  fever,  causing 
the  ioeal  reaction.  Thus  neither  the  jbcal  nor  the  general  reaction 
is  due  to  the  direct  toxic  action  of  the  tuberculin  itself.  In  this 
the  theory  approaches  somewhat  to  Marmorek's,  and  is  in  funda- 
mental opposition  to  the  older  theories  of  "addition." 

YVassermann  and  Briick  bring  forward  an  important  piece  of 
evidence  in  favour  of  their  theory  by  finding  antituberculin  present 
in  the  serum  of  patients  who  had  been  treated  with  increasing 
doses  of  tuberculin  and  had  lost  their  power  of  reacting.  In  them 
the  tuberculin  injected  would  be  immediately  neutralized  in  the 
blood,  and  so  never  reach  the  lesion.  The  theory  is  ingenious, 
and  may  possibly  turn  out  to  be  the  correct  one,  but  there  are 
difficulties.  Thus  the  authors  find  tuberculin  as  well  as  antituber- 
culin in  the  diseased  tissues,  and  it  is  difficult  to  see  why  the  two 
do  not  neutralize  one  another.  And  we  might  also  ask  why  no 
digestive  phenomena  should  follow  the  union  of  the  antituberculin 
and  tuberculin  in  the  blood  of  injected  patients,  and  the  subsequent 
absorption  of  the  complements.  Why  should  not  the  proteid 
molecules  be  digested,  liberate  their  products,  and  produce  fever  ? 
It  would  seem  that  the  antituberculin  present  in  the  lesion  must 
be  in  a  state  of  fixation  to  the  cells,  or  it  must  be  carried  away  in 
the  blood-stream,  and  this,  according  to  Wassermann  and  Briick) 
rarely  happens  except  after  injections.  But  we  do  not  know 
definitely  of  any  such  antitoxin,  the  nearest  approach  to  it  being  a 
superabundance  of  suitable  sessile  receptors,  which,  if  they  occurred, 
might  very  well  make  their  way  into  the  extracts  used  in  the  test, 
and  simulate  an  antitoxin.  And  if  this  were  the  case,  there  is  no 
explanation  why  these  receptors  are  not  shed  in  the  normal  tuber- 
culous process,  but  are  after  the  use  of  tuberculin.  It  is  difficult, 
too,  to  see  why  the  presence  of  these  abnormally  numerous 
receptors  might  not  be  made  the  basis  for  a  "  theory  of  addition  " 

20 — 2 


308       EXPLANATIONS    OF   THE    TUBERCULIN    REACTION 

without  invoking  the  aid  of  the  complements.  But  the  whole 
subject  is  theoretical  to  a  degree,  and  needs,  moreover,  independent 
experimental  verification. 

Bail's  researches  on  the  aggressins  have  been  referred  to  already. 
The  application  of  his  theory  to  Koch's  phenomenon  is  obvious. 
According  to  him  the  endotoxins  are  only  set  at  liberty  when 
bacteriolysis  occurs,  not  after  phagocytosis.  This  is  in  all  prob- 
ability correct  in  most  cases,  though  perhaps  not  in  all.  When, 
therefore,  tubercle  bacilli  are  injected  into  the  peritoneal  cavity  of 
a  normal  guinea-pig  and  extensive  phagocytosis  occurs,  there  is 
little  or  no  febrile  reaction  ;  but  in  the  tuberculous  animal  the 
bacilli  produce  aggressins,  which  paralyze  the  phagocytes,  and, 
wrhen  a  second  injection  is  made,  the  bacteria  undergo  rapid 
bacteriolysis,  endotoxin  is  set  free,  and  rapid  death  follows.  Bail 
compares  the  results  of  the  solution  of  large  quantities  of  cholera 
bacilli  in  an  immunized  animal  with  what  is  seen  after  the  injec- 
tion of  tubercle  bacilli  along  with  a  small  amount  of  the  peritoneal 
exudate  from  a  tuberculous  animal.  In  each  case  there  is  extra- 
cellular bacteriolysis,  and  death  in  a  few  hours,  obviously  from  the 
toxin  set  free. 

This  might  account  in  a  satisfactory  way  for  Koch's  phenomena 
when  caused  by  the  injection  of  cultures,  but  seems  to  fail  utterly 
when  applied  to  the  tuberculin  reaction ;  for  tuberculin  is  neither 
an  "  aggressin  "  in  Bail's  sense,  nor  an  endotoxin  of  the  tubercle 
bacillus,  and  it  cannot  undergo  bacteriolysis.  The  theory  resembles 
that  of  Marmorek. 

Von  Pirquet's  explanation  of  the  early  reaction  after  Jennerian 
vaccination  calls  for  some  notice,  though  it  is  not  immediately 
applicable  to  Koch's  phenomenon.  It  introduces  some  new  and 
interesting  conceptions.  According  to  this  author,  the  result  of 
an  infection  is  to  alter  the  way  in  which  an  animal  reacts  subse- 
quently to  a  second  infection  with  the  same  organism.  This  he 
calls  allergia.  In  some  cases  this  may  lead  to  hypersensitiveness, 
but  in  the  majority  it  leads  to  a  temporary  immunity,  followed  by 
a  condition  in  which  the  animal  is  no  longer  immune,  but  possesses 
the  power  of  forming  antibodies  in  the  region  of  inoculation  more 
quickly  and  easily  than  a  normal  one  can  do.  When  a  second 
inoculation  is  made,  the  bacteriolysins  present  in  the  blood  may  be 
sufficient  to  destroy  the  bacteria  introduced,  setting  free  their 
toxins,  which  act  locally  and  cause  the  early  reaction.  Or  this 
may  be  delayed  until  local  antibodies  are  formed.  This  occurs 


"REACTIONS"  AND  SIMILAR  PHENOMENA  309 

more  quickly  than  in  the  normal  person.  It  leads  to  an  early 
development  of  the  specific  lesion  of  vaccinia. 

The  essential  point  of  this  theory  is  that  an  infection  from  which 
recovery  has  taken  place  may  lead  to  an  alteration  of  the  facilities 
with  which  antibodies  may  be  formed,  which  alteration  persists 
for  a  long  time. 

It  seems  desirable  here  to  make  a  further  reference  to  the 
subject  already  mentioned  briefly  as  "  hypersensitiveness  to 
toxins,"  but  now  more  generally  termed  anaphylaxis— i.e.,  the 
opposite  of  prophylaxis.  The  term  was  introduced  by  Richet, 
who  studied  especially  the  poison  of  the  actiniae,  which  he  found 
to  be  extremely  powerful,  the  lethal  dose  being  about  -009  gramme 
per  kilo  of  body-weight.  He  found  that  a  non-lethal  dose  increased 
the  susceptibility  of  the  animal  to  a  second  injection,  and  that  this 
hypersensitiveness  might  last  as  long  as  six  months  after  the  first 
injection.  This,  of  course,  is  quite  similar  to  the  phenomena  we 
have  described  in  connection  with  diphtheria  and  tetanus,  which 
renders  it  so  difficult  to  immunize  small  animals  to  these  sub- 
stances, and  which  is  the  cause  of  much  danger  in  the  early  stages 
of  antitoxin  formation  in  the  higher  animals.  Richet  has  also 
studied  the  poison  formed  by  the  common  mussel,  which  he  calls 
"  mytilo-congestine,"  and  finds  exactly  similar  facts;  indeed,  it  is 
probable  that  it  is  a  general  phenomenon  of  all  the  poisons  which 
can  act  as  antigens.  In  the  case  of  mytilo-congestine  the  measure 
of  the  hypersensitiveness  is  simple,  since  one  of  the  most  constant 
symptoms  of  its  action  is  vomiting,  which  occur?  almost  as  soon 
as  the  injection  is  made.  He  finds  that  in  an  animal  which  has 
previously  been  injected  the  emetizing  dose  is  from  a  tenth  to  a 
quarter  of  the  amount  originally  necessary.  Richet  has  elaborated 
a  theory  to  account  for  this  phenomenon,  and  for  anaphylaxis  in 
general.  He  holds  that  the  condition  is  due  to  the  presence  in  the 
blood  of  a  toxogenic  substance,  which  gives  rise  to  a  poison  after 
reacting  with  the  mytilo-congestine  injected.  This  toxogenic  sub- 
stance is  not  formed  immediately,  for  Richet  does  not  find  hyper- 
sensitiveness to  come  on  for  five  or  six  days,  and  it  persists  for 
some  fifty  days,  that  being  the  average  duration  of  the  state.  He 
holds  that  the  animal  produces  antitoxin  also,  but  more  slowly. 
When  the  toxogenic  substance  has  disappeared  the  antitoxin 
remains,  and  the  animal  is  immune.  The  main  evidence  in  favour 
of  this  theory  is  the  fact  that  the  serum  of  an  anaphylactic  animal 
will  produce  a  similar  condition  in  a  second  animal.  Currie  has 


3IO  HYPERSENSITIVENESS    OR   ANAPHYLAXIS 

enunciated  a  theory  very  like  this  in  regard  to  serum  anaphylaxis, 
to  be  described  shortly. 

Other  theories  might  be  cited,  but  there  is  only  one  which  gives 
an  explanation  which  is  at  all  satisfactory  without  introducing  many 
unproved  suggestions.  It  was  introduced  quite  recently  by  Good- 
man, and  proceeds  on  lines  somewhat  similar  to  those  we  followed 
when  dealing  with  the  question  of  immunity  to  toxins.  The  cells 
of  the  body  maybe  classified  into  three  groups:  (i)  The  nerve 
cells  essential  to  life,  and  with  a  high  degree  of  affinity  for  toxin  ; 
(2)  cells  not  essential  to  life,  but  with  a  higher  degree  of  affinity 
for  toxin  than  the  nerve  cells  possess ;  and  (3)  inert  cells  without 
susceptibility  to  toxin.  If  a  dose  of  toxin  be  injected,  the  second 
class  of  cell  will  have  its  receptors  satisfied  first,  and  any  toxin 
which  is  left  over  will  then  attack  the  nerve  cells,  which  we 
assume  to  be  the  only  region  where  it  will  do  harm.  A  lethal  dose 
of  toxin,  therefore,  is  the  amount  which  will  satisfy  the  receptors 
of  the  second  group  of  cells  and  leave  enough  toxin  to  injure  the 
nerve  cells  sufficiently  to  cause  death.  Now  if  a  first  injection 
just  sufficient  to  combine  with  the  receptors  of  Group  2  were 
given,  a  very  small  additional  amount  would  be  sufficient  to  cause 
death,  since  it  would  go  straight  to  the  nerve  centres.  So  far  the 
theory  is  unsatisfactory,  since  it  is  simply  a  theory  of  summation, 
and  the  total  amount  necessary  to  cause  death,  if  given  in  divided 
doses,  should  together  form  the  amount  necessary  if  given  in  one 
dose,  which  is  very  far  from  being  the  case.  We  have  seen  that 
TJ_  of  the  "  lethal  dose  "  of  tetanus  toxin  may  cause  death  if  given 
in  divided  doses.  To  account  for  this  Goodman  supposes  that  the 
toxin  which  combines  with  the  non-essential  cells  may  cause  a  sort 
of  spreading  necrosis  of  the  receptors  of  the  latter,  or  may  interfere 
with  their  nutrition ;  in  either  case  more  of  these  receptors  may 
be  destroyed  than  the  toxin  actually  combines  with.  If  we  can 
imagine  one  molecule  of  toxin  destroying  ten  receptors,  the  animal 
would  become  as  susceptible  as  if  ten  times  the  dose  were  given 
at  once.  Put  in  another  way,  if  it  takes  x  molecules  of  toxin  to 
satisfy  the  receptors  of  non-essential  cells,  and  }(  molecules  to 
combine  with  those  of  the  central  nervous  system  and  kill,  then 
if  in  the  sensitizing  dose  each  molecule  of  toxin  destroys  ten 
receptors,  the  lethal  amount  necessary  for  a  second  dose  would  be 

but  —  +  a.     x  we  must  suppose  much  larger  than  a. 

He   compares   this   process  with  the  injury  to  the  excretory 


"REACTIONS"  AND  SIMILAR  PHENOMENA  311 

organs  which  often  follows  the  action  of  poison.  The  kidneys, 
etc.,  excrete  the  substance,  but  in  doing  so  are  injured,  and  a 
smaller  dose  of  the  poison  may  now  produce  a  great  effect,  since 
it  cannot  readily  be  eliminated. 

The  main  objection  to  this  theory  is  that  it  is  difficult  to 
imagine  such  a  selective  destruction  of  the  receptors  as  seems 
necessary  to  account  for  the  fact  that  the  hypersensitiveness  is 
specific.  We  should  expect  the  creeping  necrosis  or  interference 
with  nutrition  to  act  more  generally,  so  that  an  animal  highly 
sensitized  to  one  toxin  would  show  some  degree  of  sensitiveness 
to  others.  It  seems  also  inadequate  to  explain  the  facts  of  serum 
anaphylaxis,  which  will  now  be  described,  since  here  the  animal 
is  sensitized  with  minute  amounts  of  a  substance  which  causes  no 
toxic  symptoms  in  comparatively  enormous  doses  in  a  normal 
animal.  Here  the  anaphylaxis  appears  to  be  the  production  of  a 
new  sensitiveness  rather  than  the  exaltation  of  one  previously 
existing. 

There  are  two  of  these  phenomena  of  hypersensitiveness  to 
serum — Arthus'  phenomenon  and  Theobald  Smith's  phenomenon, 
both  of  which  are  referred  to  as  "  serum  anaphylaxis."  The  latter 
is  the  more  important. 

Arthus'  phenomenon  appears  when  a  guinea-pig  receives 
several  injections,  at  intervals  of  a  few  days,  of  normal  horse 
serum,  a  substance  which  in  itself  is  scarcely  more  toxic  than 
normal  solution.  After  a  few  such  inoculations  the  animal  becomes 
hypersensitive,  or  anaphylactized,  and  after  another  injection  an 
cedematous  mass,  an  aseptic  abscess,  or  an  area  of  necrosis, 
appears  at  the  site  of  a  new  inoculation,  which  need  not  be  in  a 
region  in  which  a  previous  injection  has  been  made ;  the  altera- 
tion is  a  general,  and  not  a  local,  one.  After  several  of  these 
injections  the  animal  becomes  cachectic,  and  dies  after  several 
weeks.  An  animal  thus  sensitized  will  die  rapidly  after  the 
injection  of  2  c.c.  of  serum  into  the  veins. 

It  should  be  noticed  that  these  results  are  not  due  to  the  accu- 
mulation of  the  horse  serum  in  the  system,  since  they  may  be 
brought  about  by  the  injection  in  divided  doses  of  an  amount 
which  an  animal  can  stand  with  impunity  if  given  in  a  single 
dose. 

Theobald  Smith's  phenomenon  occurs  when  an  animal  has  been 
sensitized  by  a  very  small  injection  of  horse  serum  (TJ^  c.c.,  or 
even  as  little  as  T^nro^nnr  c>Ct'  an  almost  inconceivably  small 


312  THEOBALD    SMITH'S    PHENOMENON 

amount  to  produce  so  great  an  effect),  and  kept  for  a  fortnight  or 
more.  If  then  a  second  injection  of  a  larger  amount  of  the  same 
serum  be  made  (^  c.c.  or  more,  the  usual  testing  dose  being  5  c.c.), 
the  animal  develops  a  series  of  remarkable  symptoms,  the  most 
noteworthy  being  respiratory  failure,  paralysis,  and  clonic  spasms. 
Symptoms  usually  appear  within  ten  minutes,  and  death  occurs 
within  an  hour.  Death  does  not  always  follow.  The  less  sensitive 
the  animal,  the  later  the  development  of  symptoms  (which  in 
highly  sensitive  animals  come  on  within  ten  minutes),  and  the 
greater  the  chance  of  survival.  The  process  evidently  affects  the 
nervous  system  in  a  very  special  way,  and  the  heart  may  continue 
to  beat  for  an  hour  after  death.  In  some  cases,  but  not  in  all, 
there  are  definite  haemorrhagic  lesions  present ;  they  usually  occur 
in  the  stomach,  less  frequently  in  the  caecum,  lungs,  spleen, 
adrenals,  or  other  parts. 

The  phenomena  had  often  been  seen  in  the  process  of  testing 
diphtheria  and  other  antitoxins  for  the  presence  of  free  toxin,  in 
which  several  cubic  centimetres  of  the  serum  are  injected  intra- 
peritoneally  into  guinea-pigs.  Animals  that  have  been  previously 
used  for  the  standardization  of  the  antitoxin  are  often  employed, 
and  as  these  have  received  minute  doses  of  the  latter  substance 
they  may  be  hypersensitive.  The  phenomenon  is  a  familiar  one, 
but  it  is  only  recently  that  its  true  method  of  origin  has  been 
apparent.  It  has  no  connection  with  the  antitoxin  as  such,  and 
the  same  phenomena  of  hypersensitiveness  may  be  produced  by 
means  of  egg-albumin. 

The  action  is  to  a  certain  extent  a  specific  one.  An  animal 
sensitized  with  horse  serum  is  less  susceptible  to  the  serum  of  the 
cow,  pig,  sheep,  etc.,  than  to  that  of  the  horse.  It  may  show 
symptoms  after  the  injection  of  one  of  these  heterologous  sera, 
but  usually  recovers.  And  the  same  is  true  .for  an  animal  sensi- 
tized by  small  doses  of  another  sera.  Symptoms  are  not  usually 
produced  by  horse  serum,  and  if  they  are,  are  not  fatal.  Animals 
can  be  sensitized  by  feeding  with  horse  serum  or  with  horseflesh. 
Rosenau  and  Anderson  thought  that  children  might  be  sensitized 
in  this  way,  and  so  develop  toxic  symptoms  after  the  use  of  anti- 
toxin, but  abandoned  the  idea. 

Otto  and  Rosenau  and  Anderson  thought  that  small  doses  were 
necessary  for  the  production  of  this  form  of  hypersensitiveness, 
large  ones  appearing  to  bring  about  immunity ;  but  Gay  and 
Southard  show  that  large  doses  simply  delay  the  incubation 


"  REACTIONS  "    AND    SIMILAR    PHENOMENA  313 


period.  After  an  injection  of  TJ^  c.c.  or  Ti)ir  c.c.  the  animal  is 
hypersensitive  in  a  fortnight  or  less,  whereas  after  a  dose  of  8  c.c. 
the  sensitiveness  does  not  reach  its  maximum  for  some  forty-five 
days.  The  duration  of  this  anaphylaxis  is  not  exactly  determined, 
but  it  certainly  lasts  several  months. 

Gay  and  Southard  further  found  that  during  the  period  of  in- 
sensitiveness  which  follows  a  large  dose  the  animal  actually 
contains  the  substance  which  acts  as  a  sensitizing  agent.  Thus  a 
guinea-pig  which  had  received  (in  divided  doses)  17  c.c.  of  normal 
horse  serum  was  bled  fourteen  days  after  the  last  dose  :  1*5  c.c. 
of  its  serum  was  found  to  sensitize  a  normal  guinea-pig,  so  that  it 
died  in  ninety  minutes  after  an  injection  of  normal  horse  serum. 
(Rosenau  and  Anderson  had  already  found  that  the  young  of 
sensitized  animals  are  also  sensitive.)  Further,  Gay  and  Southard 
found  the  sensitizing  substance  present  in  the  blood  of  sensitized 
animals.  Thus  a  guinea-pig  received  TJ<j-  c.c.  of  horse  serum, 
and  after  twenty-nine  days  was  bled,  and  1-5  c.c.  found  to  sensi- 
tize another  animal.  The  first  pig  was  tested  and  found  to  be 
sensitive. 

These  discoveries  are  sufficiently  astonishing,  and  there  appears 
to  be  no  satisfactory  explanation  for  them.  The  period  of  incuba- 
tion would  suggest  that  we  are  dealing  with  antibodies  of  some 
sort,  but  there  is  no  evidence  to  show  that  the  precipitins  play  any 
part  in  the  process.  An  animal  may  be  highly  sensitive  when  no 
precipitin  can  be  demonstrated  in  its  serum. 

Gay  and  Southard  think  that  the  hypersensitiveness  depends  on 
the  presence  of  a  substance  which  occurs  in  horse  serum,  and 
which  they  distinguish  by  the  name  anaphylactin.1  This  they  do 
not  consider  to  be  the  same  as  the  toxic  ingredient  of  the  serum, 
for  this  reason :  a  sensitized  animal  will  not  develop  symptoms 
after  the  injection  of  blood  from  an  animal  in  the  refractory  stage, 
though  this,  as  we  have  seen,  was  sufficient  to  sensitize  it.  (The 
injections  were  made  into  a  vein,  the  sensitive  animal  reacting  to 
very  minute  doses  administered  by  this  channel.)  They  failed  to 
find  any  proof  of  the  formation  of  antibodies  in  the  animal  during 
the  refractory  stage.  They  regard  the  reaction  as  being  one  of 
the  cells  of  the  sensitized  animal,  and  as  being  due  to  a  heightened 
power  of  absorption  of  the  "  toxic  substance  "  on  the  part  of  cells 
which  have  been  exposed  for  a  certain  period  to  the  action  of  the 
anaphylactic  substance.  The  action  of  this  toxic  substance 
1  Analogous  to  Richet's  toxogenic  substance. 


314  THEOBALD    SMITH'S    PHENOMENON 

appears  to  be  to  make  the  fatty  constituents  of  the  cells  flow 
rapidly  together.  This  occurs  especially  in  the  endothelium  of 
the  capillaries,  and  leads  to  haemorrhages. 

This  can  hardly  be  regarded  as  a  full  explanation,  and  fails, 
moreover,  to  explain  a  most  remarkable  fact  discovered  by 
Besredka  :  that  sensitized  animals  do  not  react  to  the  injection  of 
horse  serum  if  previously  anaesthetized  with  ether.  No  theory 
that  has  yet  been  suggested  will  explain  all  the  extraordinary 
phenomena  connected  with  this  subject,  and  no  good  purpose 
would  be  served  by  discussing  others  that  have  been  brought 
forward.  We  seem  to  be  on  the  eve  of  a  series  of  discoveries 
that  may  prove  to  have  as  great  an  effect  on  our  ideas  of  immunity 
and  cell  nutrition  as  the  discovery  of  the  antibodies  themselves, 
and  until  the  facts  are  better  known  hypotheses  seem  out  of 
place. 

We  may,  however,  be  permitted  to  point  out  that  it  is  quite 
possible  that  the  tuberculin  reaction  may  be  a  phenomenon  of 
exactly  the  same  order  as  the  serum  anaphylaxis  of  the  guinea-pig. 
Tuberculin  is  in  itself  non-toxic  for  a  normal  animal,  just  as  is 
serum,  but  we  may  imagine  that  the  tubercle  bacilli  in  the  lesions 
produce  it  in  small  amounts  until  the  animal  becomes  anaphy- 
lactized,  in  which  case  it  will  react  violently  to  a  small  injection. 
The  wasting  and  fever  which  accompany  the  evolution  of  the 
disease  may  be  phenomena  akin  to  the  phenomenon  of  Arthus, 
and  simply  indicate  the  reaction  of  a  hypersensitive  animal  to 
repeated  small  doses  of  a  non-toxic  substance.  If  this  is  the  case 
we  may  regard  tuberculin  as  being,  after  all,  the  true  toxin  of  the 
disease,  though  non-toxic  to  animals  unless  the  processes  of  tissue 
nutrition  and  metabolism  have  been  profoundly  modified  by  the 
prolonged  action  of  minute  amounts  of  the  same  substance.  That 
a  reaction  does  not  occur  in  a  normal  person  after  two  injections 
of  tuberculin  does  not  surprise  us,  for  the  active  principle  is  (as 
shown  by  the  fact  that  it  dialyzes)  of  small  molecule,  and  may  be 
eliminated  more  quickly  than  the  large-moleculed  toxic  ingredient 
of  horse  serum,  so  that  for  hypersensitiveness  to  occur  it  may  be 
necessary  to  have  a  continued  stream  of  minute  amounts  from  a 
lesion  in  the  tissues.  It  may  be  pointed  out,  however,  that  occa- 
sionally a  local  reaction  may  be  seen  round  the  spots  at  which 
tuberculin  has  been  previously  injected,  suggesting  that  the  tissues 
in  this  region  may  be  anaphylactic. 

There  appears  to  be  no  connection  between  this  phenomenon  of 


"  REACTIONS  "    AND    SIMILAR    PHENOMENA  315 

serum  anaphylaxis  and  the  few  cases  that  have  occurred  of  sudden 
death  after  the  injection  of  antitoxin,  since  these  have  usually  (not 
invariably)  occurred  after  the  first  dose.  In  some  cases,  if  not 
in  all,  they  have  happened  in  children  the  subjects  of  the  lymphatic 
diathesis,  who  are  subject  to  sudden  death  on  very  slight  provoca- 
tion. And  the  remarkable  lesions  recorded  by  Gay  and  Southard 
have  never  been  seen  after  death  from  a  single  dose  of  antitoxin ; 
nor  do  the  extraordinary  symptoms  develop. 

This  seems  the  most  suitable  place  in  which  to  discuss  the 
"  serum  disease,"  or  series  of  unpleasant  though  transient  pheno- 
mena which  may  occur  after  the  use  of  antitoxin,  though  it  must 
not  be  considered  that  it  is  necessarily  a  phenomenon  having  any 
connection  with  those  that  have  been  already  described. 

The  symptoms  have  been  carefully  analyzed  by  von  Pirquet, 
and  the  following  account  is  based  very  largely  on  his  observations. 
The  most  constant  symptom  is  fever,  which  is  usually  remittent 
in  type.  Next  in  frequency  is  a  rash,  which  may  be  general  or 
confined  to  the  vicinity  of  the  region  at  which  the  injection  was 
made.  The  type  of  the  rash  varies  in  different  cases,  but  all 
forms  are  associated  with  the  most  unpleasant  symptom  of  the 
serum  disease — namely,  severe  itching.  The  types  of  rashes  are 
those  included  under  the  term  "erythema  multiforme."  The 
severest,  which  is  associated  with  the  greatest  degree  of  fever, 
is  the  morbilliform  ;  the  scarlatiniform  is  associated  with  less  ; 
and  the  urticarial  rash,  which  is  the  commonest,  is  usually  accom- 
panied with  but  little  elevation  of  temperature. 

There  is  usually  enlargement  of  the  lymphatic  glands  corre- 
sponding to  the  region  where  the  injection  was  administered,  and 
this  enlargement  may  be  the  first  sign  of  the  onset  and  of  the  cure 
of  the  disease.  The  pains  in  the  joints  often  form  one  of  the  most 
unpleasant  of  the  symptoms,  and  the  articulations  usually  affected 
are  the  metacarpo-phalangeal,  wrist,  and  knee,  in  that  order  of 
frequency.  Albuminuria,  not  going  on  to  nephritis,  is  occasionally 
present,  and  slight  oedema  is  common.  The  severity  of  the 
disease  is  roughly  proportioned  to  the  amount  of  serum  injected. 

According  to  von  Pirquet  and  Schick  the  leucocytes  are 
increased  in  number  until  the  symptoms  develop,  when  a  rapid 
fall  (due  chiefly  to  a  decrease  in  the  polynuclears)  takes  place. 

The  disease  is  painful,  but  not  dangerous,  complete  recovery 
invariably  occurring.  The  few  cases  of  sudden  death  that  have 
been  recorded  as  taking  place  after  an  injection  of  serum  appear 


3*6  THE 

to  be  quite  different  in  nature,  and  in  many  cases  were  dependent 
on  the  "  status  lymphaticus,"  or  "  thymicus,"  a  condition  in 
which  a  slight  disturbance  of  any  kind  may  cause  sudden 
death. 

In  general  terms,  the  severity  and  frequency  of  the  serum 
disease  are  proportionate  to  the  amount  of  serum  given  —  the  larger 
the  dose,  the  greater  the  likelihood  of  its  development  and  the  more 
severe  the  disease.  There  are,  however,  some  exceptions  : 
(i)  Certain  samples  of  serum  appear  more  potent  in  this  respect 
than  others.  The  purified  diphtheria  antitoxin  prepared  by 
Gibson  (which  consists  of  a  solution  of  globulin)  appears  to 
reduce  the  occurrence  of  the  disease  to  a  minimum.  (2)  The 
serum  disease  is  more  likely  to  occur  when  a  second  injection  is 
given  at  an  interval  of  three  or  four  weeks  or  longer  after  the 
first. 

Under  ordinary  circumstances  the  disease  manifests  itself  after 
an  interval  of  eight  to  twelve  days,  or  sometimes  longer,  after  the 
injections.     This   period    is    insignificant,  since   it   approximates 
closely  to  the  period  of  maximum  development  of  most  of  the 
antibodies   after    an    injection    of    an    antigen.       Hence    it    was 
suggested  (by  Hamburger  and  others)  that  the  symptoms  might 
be  due  to  the  development  of  a  precipitin,  which,  by  combining 
wTith  the  unaltered  horse  serum  still  present  in  the  patient's  blood, 
might  cause  the  production  of  small  precipitates  in  the  circula- 
tion.    These,  being  deposited   in   the  minute  capillaries   of   the 
skin,  joints,  etc.,  might  act  as  emboli,  and  cause  the  characteristic 
symptoms.      Now  the  blood  of  a  patient  who  has  been  treated 
with  serum  is  frequently  found  to  contain  precipitin  after  a  week 
or  ten  days,  so  that  the  possibility  of  this  explanation  is  obvious. 
The  phenomenon  of  the  accelerated  reaction  also  appears  to  lend 
it  support.     A  patient  who  has  been  injected  with  horse  serum 
may  be  presumed  to  have  precipitin  persisting  in  his  system  for 
some  unknown  but  possibly  lengthy  period  afterward,  and  on  the 
injection  of  more  horse  serum  (its  antigen)  might  cause  a  precipi- 
tate which  would  lead  to  an  immediate  or  accelerated  development 
of  the  serum  disease.     It  is  found  in  practice  that  this  immediate 
reaction  does  occur,  but  is  comparatively  rare.     In  ninety  cases 
where  a  second  injection  was  given  the  disease  was  developed 
within  six  hours  in  nine  ;  in  thirty-nine  others  it  was  produced 
within  a  period  varying  from  nineteen  hours  to  five  days  (Goodall). 
The  "  immediate  effect"  described  by  Goodall  differs  somewhat 


"REACTIONS"  AND  SIMILAR  PHENOMENA  317 

from  the  usual  "  accelerated  effect "  occurring  in  patients  injected 
with  serum  for  the  second  time.  The  symptoms  are  rigor, 
pyrexia,  vomiting,  rash,  and  collapse,  whereas  the  effects  which 
develop  in  a  day  or  two  consist  of  rash,  joint  pains,  and  pyrexia. 

The  precipitation  theory  is  now  generally  abandoned,  since 
further  investigation  shows  that  there  is  no  close  parallelism 
between  the  occurrence  of  the  disease  and  the  presence  of 
precipitins  in  the  serum.  Thus  it  is  often  found  that  the  symptoms 
are  present  when  not  the  slightest  trace  of  antihorse  precipitin  is 
demonstrable.  On  the  other  hand,  precipitins  may  occur  when 
the  disease  does  not  develop,  though  this  is  a  fact  of  less  impor- 
tance, since  it  is  necessary,  on  this  theory,  that  the  precipitin  and 
the  unaltered  horse  serum  should  be  present  simultaneously,  and 
this  latter  fact  is  usually  impossible  of  proof.  But  Widal  and 
Rostane  have  brought  forward  very  strong  negative  evidence  in 
showing  that  the  disease  does  not  necessarily  occur  after  the 
intravenous  injection  of  a  powerful  antihuman  precipitating 
serum  in  man. 

These  arguments,  though  strong,  cannot  be  regarded  as  entirely 
conclusive.  As  regards  the  absence  of  the  precipitin  in  some 
cases  of  serum  disease,  it  may  be  pointed  out  (a)  that  it  may  have 
all  been  removed  in  the  form  of  precipitum  when  the  sample  was 
collected,  and  (b)  that  the  demonstration  in  vitro  of  a  minute 
amount  of  precipitin  is  by  no  means  easy,  and  requires  an  appro- 
priate correlation  between  the  precipitating  and  normal  sera,  or 
the  phenomenon  may  be  missed.  Further — and  this  applies  to  the 
negative  experiments  of  Widal  and  Rostane — we  do  not  know 
exactly  what  conditions  are  necessary  to  the  union  of  precipitin 
and  antigen  to  form  an  insoluble  precipitate,  and  whether  these 
are  always  present  in  vivo.  Some  experiments,  it  is  true,  appear 
to  show  that  the  combination  never  occurs  in  the  animal  body, 
but  they  are  inconclusive,  and  the  supposition  is  in  the  highest 
degree  unlikely.  It  appears  probable  that  precipitation,  like 
agglutination,  depends  on  the  presence  of  certain  salts,  as  well  as 
on  that  of  the  antibody  and  antigen,  and  it  may  be  that  these  are 
sometimes  absent  or  not  present  in  an  available  form.  In  this 
connection  we  may  quote  the  researches  of  Netter,  which  are  of 
great  practical  importance.  He  showed  that  the  administration 
of  calcium  lactate  at  the  time  of  the  injection,  and  for  a  day  or 
two  after,  caused  a  great  diminution  in  the  number  of  cases  of  the 
serum  disease.  His  results  have  been  generally  substantiated, 


318  THE 

and,  in  addition,  the  drug  has  been  found  to  be  of  decided 
therapeutic  value.  Hence  it  appears  that  a  diminished  calcium 
content  of  the  patient's  blood  at  the  time  of  the  injection  may  be 
of  some  importance  in  bringing  about  the  conditions  necessary  for 
the  development  of  the  disease.  If  this  is  the  case,  it  might 
account  for  the  symptoms  in  some  cases  and  not  in  others, 
although  precipitin  might  be  produced  in  both.  And  there  are 
probably  other  factors  which  are  at  present  unsuspected,  or  the 
use  of  the  calcium  salt  would  be  efficacious  in  all  cases. 

The  alternative  theory  is,  of  course,  that  the  patient  gradually 
becomes  hypersensitive  to  the  serum,  and  that  the  grade  of 
hypersensitiveness  necessary  for  a  reaction  is  reached  before  the 
serum  is  eliminated  from  the  blood,  or,  in  the  case  of  an  im- 
mediate or  accelerated  reaction,  has  not  passed  off  before  the 
second  injection  is  given.  It  would  make  it  a  phenomenon 
analogous  to  those  of  Koch  or  Theobald  Smith. 

A  good  argument  in  favour  of  this  theory  is  the  long  interval 
which  may  occur  between  the  first  injection  and  the  second  in  the 
case  of  an  accelerated  reaction.  This  may  be  a  year  or  more — a 
very  long  period  for  antibodies  to  persist  in  the  blood  after  a  single 
injection  of  antigen. 


CHAPTER  XII 
COLLOIDAL  THEORY  OF  ANTIBODIES 

THE  fact  that  antigens  and  their  antibodies  are,  as  far  as  is  known 
at  present,  exclusively  colloid  in  chemical  character  renders  it 
advisable  to  glance  briefly  at  some  of  the  main  facts  and  theories 
concerning  these  bodies  and  their  reactions.  They  are  substances 
of  the  greatest  possible  interest  to  the  biologist,  since  the  living 
tissues  and  the  fluids  of  the  living  animal  are,  without  exception, 
colloids  and  colloidal  solutions ;  protoplasm,  cell  nuclei,  etc.,  being 
mixtures  of  colloids  in  a  jelly-like  form,  whilst  the  fluid  part  of 
blood-lymph,  etc.,  is  a  colloidal  solution.  It  is  necessary  to 
remember  this,  since  the  reactions  of  colloids  and  of  crystalloids 
in  presence  of  colloid  appear  to  follow  laws  which  are  different 
from  those  governing  the  reactions  of  crystalloids  alone,  and  we 
may  doubt  whether  these  latter  ever  take  place  in  the  living  body. 
Colloids  are  not  necessarily  organic  bodies,  since  metals  such  as 
gold  and  silver  may  be  obtained  in  the  colloidal  state,  as  well  as 
many  metallic  salts,  such  as  ferric  oxide,  silicic  acid,  etc.  These 
simple  colloids  appear  to  be  governed  by  laws  similar  to  those 
concerned  in  the  reactions  of  the  organic  colloids  formed  by  the 
action  of  vital  processes. 

The  chief  features  which  distinguish  colloids  from  crystalloids 
are — (i)  that  they  do  not  undergo  dissociation  into  their  ions  when 
dissolved  in  water ;  and  (2)  that  this  so-called  solution  is  not  a  true 
one,  but  merely  a  suspension  of  unaltered  molecules  or  groups  of 
molecules.  These  two  characters  are  probably  fundamentally  the 
same.  A  colloid  solution,  therefore,  is  merely  a  very  fine  emulsion 
or  suspension  of  particles  of  the  substance,  and  Siedentopf  and 
Zsigmondy  have  demonstrated  a  method  by  which  these  particles, 
though  infinitely  small  as  compared  with  the  most  minute  bacteria, 
can  be  rendered  visible  and  their  numbers  estimated.  The  method 
is  simple  enough  in  theory,  though  the  actual  arrangements  are 
somewhat  complicated.  A  powerful  beam  of  light  is  passed  trans- 

319 


32O         NATURE  OF  COLLOIDAL  SOLUTIONS 

versely  through  a  true  solution  placed  in  the  field  of  a  powerful 
microscope,  the  ray  passing  at  right  angles  to  the  optical  axis. 
No  light  enters  the  lens,  and  the  whole  field  remains  in  darkness. 
If,  however,  a  colloid  solution  be  similarly  treated,  these  particles 
will  reflect  the  light  in  every  direction,  and  some  rays  will  enter 
the  lens.  The  result  is  that  the  particles  are  seen  as  minute 
luminous  points  on  a  dark  background.  The  process  is  analogous 
with  the  examination  of  the  stars  with  a  telescope,  wrhich,  no 
matter  how  powerful,  never  shows  the  star  as  a  disc,  but,  merely 
by  collecting  more  light,  renders  it  brighter,  and  shows  stars  so 
small  as  to  be  invisible  to  the  naked  eye. 

The  particles  in  question  are  so  small  that  they  are  prevented 
from  falling  to  the  bottom  of  the  fluid  by  friction ;  if,  however, 
they  clump  together,  the  particles  become  heavier,  whilst  the 
surface  which  they  expose  to  the  fluid  does  not  increase  at  so  great 
a  rate,  until  at  last  aggregates  of  molecules  are  formed,  which  fall 
more  or  less  rapidly.  This  is  what  takes  place  in  coagulation  of 
a  proteid,  whether  brought  about  by  heat  or  by  the  addition  of 
electrolytes,  etc.  The  factors  which  inhibit  and  which  cause  this 
clumping  of  molecules  thus  come  to  be  of  vital  importance.  The 
force  which  tends  to  cause  this  clumping  is  probably  surface 
tension  (Hardy,  Bredig,  Perrin),  which  is  developed  at  the  junc- 
tion of  the  molecule  and  the  water,  and  which,  as  explained  in  the 
section  dealing  with  agglutination,  tends  to  draw  together  any 
particles  within  a  certain  distance  of  one  another,  so  that  the 
surface  may  become  as  small  as  possible.  This,  of  course,  is  not 
a  peculiarity  of  colloidal  solutions,  but  occurs  in  all  emulsions  of 
insoluble  particles.  Surface  tension,  therefore,  is  constantly  tend- 
ing to  make  the  particles  of  colloid  in  a  solution  approach  one 
another,  form  aggregates,  and  so  cause  precipitation  or  coagula- 
tion. 

This  action  is  counterbalanced  in  a  stable  solution  by  a  force 
of  electrical  repulsion.  The  colloid  particles  all  carry  a  feeble 
charge  of  positive  or  negative  electricity,  and  therefore  tend  to 
repel  one  another.  The  existence  of  this  charge  and  its  nature 
can  be  shown  by  passing  an  electrical  current  through  the  fluid. 
Under  these  circumstances  the  colloids  do  not  undergo  dissocia- 
tion with  passage  of  the  +  ions  to  the  -  pole,  and  vice  versa,  like 
the  electrolytes ;  instead,  the  molecules  pass  bodily  to  one  or  other 
pole,  according  to  the  sign  of  the  electric  charge  they  carry.  Thus 
Field  and  Teague  find  that  antitoxin  is  carried  towards  the  cathode, 


COLLOIDAL    THEORY    OF   ANTIBODIES  321 

and  must  therefore  carry  a  positive  charge.  Further  researches 
show  that  absolutely  pure  colloids,  free  from  all  traces  of  electro- 
lytes, carry  no  charge  at  all,  and  are  not  conveyed  by  an  electrical 
current.  The  charge  depends  upon  the  nature  of  the  ions  present : 
traces  of  acid  and  of  acid  salts  give  it  a  positive  charge,  whilst 
alkalis  and  alkaline  salts  do  the  reverse  (Pauli). 

The  process  of  coagulation  of  proteids,  therefore,  must  depend 
upon  the  neutralization  of  this  electrical  charge,  and  this  can  be 
accomplished  either  by  electrolytes  or  by  colloids.  Non-electro- 
lytes (i.e.,  substances  which  do  not  split  up  into  electrified  ions  in 
solution)  do  not  bring  about  coagulation  in  this  way.  Many  of 
them,  such  as  sugar  and  urea,  are  inert ;  others,  such  as  alcohol, 
act  in  an  entirely  different  manner.  The  precipitation  of  an  albu- 
minous solution  by  means  of  a  strong  acid  takes  place  thus :  The 
negatively-charged  particles  attract  to  themselves  the  positively- 
charged  hydrogen  ions ;  their  charge  is  now  neutralized,  and  the 
force  of  attraction  due  to  their  surface  tension  is  no  longer  counter- 
balanced by  an  electrical  repulsion ;  the  particles  are  drawn 
together,  form  larger  and  larger  aggregates,  and  finally  cohere  into 
masses  so  large  as  to  come  under  the  influence  of  gravity,  when 
precipitation  takes  place.  The  coagulation  of  albumin  by  alcohol 
is  due  to  the  fact  that  proteids  are  not  soluble  in  that  fluid,  so  that 
when  it  is  added  to  a  watery  solution  of  proteid,  the  water  is  with- 
drawn and  the  particles  brought  so  close  together  that  surface 
tension  comes  into  play,  and  makes  them  coalesce  into  aggregates. 
The  truth  of  this  explanation  appears  from  Pauli's  observation 
that  when  no  electrolytes  are  present  alcohol  acts  very  readily  as 
a  precipitant — there  is  no  electrical  repulsion  between  the  particles. 
But  if  a  little  acid  or  alkali  be  added,  and  the  molecules  be  thus 
given  a  mutually  repellent  electrical  charge,  the  precipitation  is 
inhibited  or  entirely  prevented.  Colloids  can  also  be  precipitated 
by  colloids  as  well  as  by  electrolytes,  but  in  this  case  they  must 
be  of  opposite  sign.  Thus,  in  testing  for  albumin  in  urine  by 
means  of  acetic  acid  and  ferrocyanide  of  potash,  the  addition  of 
the  acid  insures  that  the  particles  shall  acquire  a  positive  charge 
(if  they  have  it  not  already),  which  is  then  neutralized  by  the 
colloidal  ferrocyanic  acid  of  negative  sign.  Numerous  other 
examples  might  be  quoted. 

This  taking  up  of  substances  by  colloidal  particles  is  an  example 
of  the  phenomenon  known  as  adsorption.  This  process  is  difficult 
of  definition,  but  in  general  terms  we  may  regard  it  as  a  combina- 

21 


322  ADSORPTION 

tion  between  two  substances  dependent  on  physical  attraction 
rather  than  on  chemical  affinity,  and  taking  place  in  very  variable 
ratios,  rather  than  in  simple  ones  dependent  on  combinations  of 
atoms  or  molecules,  such  as  occurs  in  true  chemical  union.  But 
it  must  be  admitted  that  there  is  no  absolutely  sharp  line  of 
demarcation  between  the  two  classes  of  phenomena.  In  most 
cases  the  two  substances  entering  into  the  phenomenon  of  adsorp- 
tion exist  as  such  side  by  side  in  the  compound,  which  is  to  be 
regarded  rather  as  an  intimate  admixture  of  the  two  than  as  an 
entirely  new  substance.  The  question  arises  as  to  whether  the 
union  between  antigen  and  antibody  is  not  really  a  process  of 
adsorption,  and  in  the  attempt  to  solve  this  problem  several  very 
startling  analogies  with  colloidal  adsorption  have  been  discovered. 
It  will  be  convenient  to  discuss  some  of  these,  and  then  to  give 
an  account  of  the  analogies  met  with  in  the  chemistry  of  the 
colloids. 

Bordet  has  shown  that  the  amount  of  haemolytic  immune  body 
which  can  be  taken  up  by  a  given  volume  of  corpuscles  varies 
according  to  whether  the  corpuscles  be  added  at  the  same  time  or 
in  successive  small  portions.  Thus,  in  one  example  0-4  c.c.  of  a 
haemolytic  serum  dissolved  0-5  c.c.  of  corpuscles  if  added  at  once. 
But  if  0-2  c.c.  of  corpuscles  were  added  first,  and  then  successive 
amounts  of  0*1  c.c.  put  in,  no  solution  took  place  after  that  of  the 
first  portion  added.  This  he  explains,  as  we  have  already  seen,  by 
invoking  a  physical  process  of  the  nature  of  adsorption,  comparing 
it  with  the  adsorption  of  a  dye  by  filter-paper.  Other  explanations 
may  be  possible,  but  an  exactly  similar  phenomenon  may  be  seen 
in  the  mutual  adsorption  of  colloids  of  opposite  sign.  Thus,  if  to 
a  given  amount  of  solution  of  an  electro-positive  colloid  an  amount 
of  solution  of  an  electro-negative  colloid  exactly  sufficient  for 
neutralization  of  the  opposite  charges  be  added,  the  result  will  be 
the  immediate  commencement  of  the  process  of  agglutination, 
which  will  go  on  until  all  the  colloids  are  precipitated.  But 
if  a  small  amount  of  the  second  colloid  be  added  to  the  same 
volume  of  the  other,  a  different  state  of  affairs  is  brought  about- 
New  aggregates  of  the  two  are  formed,  which  are  not  necessarily 
neutral  in  reaction,  and  experience  shows  that  the  conditions  are 
now  less  favourable  to  precip  tation,  which  may  require  much 
more  of  the  second  colloid  for  its  complete  accomplishment,  the 
intermediate  bodies  being  apparently  less  easily  precipitated  than 
the  unaltered  colloid.  Similar  phenomena  may  also  be  seen  in  the 


COLLOIDAL    THEORY    OF   ANTIBODIES  323 

action  of  agglutinin  on  bacteria.  Here  also  the  solid  particles  may 
take  up  much  more  antibody  than  is  necessary  for  agglutination 
to  be  induced  if  the  serum  be  added  all  at  once.  It  may  be  pointed 
out  that  this  has  a  practical  value  in  the  estimation  of  the  degree 
of  agglutinating  action  as  determined  by  the  degree  of  dilution  in 
which  the  action  will  take  place.  It  is  not  proper  to  make  a  strong 
dilution  of  the  serum  in  the  bacterial  emulsion,  and  to  dilute  this 
with  further  amounts  of  the  latter,  unless  the  subsequent  dilutions 
are  made  quickly.  Otherwise,  all  the  agglutinin  may  be  removed 
by  the  bacilli  in  the  first  dilutions,  and  those  subsequently  added 
be  apparently  unaltered.  The  most  accurate  method  is  to  prepare 
all  the  serum  dilutions  required  of  double  strength,  and  to  add  to 
each  an  equal  volume  of  bacterial  emulsion.  On  the  other  hand, 
when  a  bacterial  emulsion  is  added  to  a  serum  in  successive  doses, 
more  agglutinin  is  taken  up  than  when  the  whole  amount  is  added 
at  once. 

Next  as  regards  the  phenomena  in  which  an  excess  of  antibody 
has  apparently  a  reversing  action,  the  best-known  examples  of 
which  are  :  (i)  the  deviation  of  complement,  or  Neisser-Wechsberg 
phenomenon ;  (2)  the  presence  of  zones  of  inhibition  in  which  no 
precipitation  occurs  in  mixtures  of  serum  and  precipitin ;  and 
(3)  the  similar  phenomena  observable  in  the  agglutinins.  All 
these  have  been  discussed  previously  and  explanations  suggested. 
These  explanations  have  the  merit  of  not  involving  any  pheno- 
mena of  nature  different  to  those  familiar  in  immunity  reactions, 
but  there  are  objections  to  all.  Thus,  Neisser  and  Wechsberg's 
explanation  of  the  deviation  of  complement  would  have  us  believe 

CLfiH^no  C  C  fJ  f-p-r- 

that  uncombined  complement  has  a  greater  affinity  for  alexin  than 
that  which  has  had  its  cytophile  groups  combined  with  cell  re- 
ceptors— a  phenomenon  exactly  opposite  to  that  which  occurs  in 
haemolysis,  where  the  process  can  be  studied  with  greater  accuracy. 
We  have  also  seen  difficulties  in  the  way  of  accepting  Gay's 
explanation,  the  full  particulars  of  which  are  not  yet  available. 
In  the  same  way  the  inhibiting  effects  of  large  doses  of  precipi- 
tating or  agglutinating  serum  may  apparently  be  explained  on  the 
hypothesis  of  the  existence  of  precipitoids  and  agglutinoids  of 
higher  combining  affinity  than  their  unaltered  antibodies,  but  it  is 
at  least  doubtful  whether  this  is  entirely  satisfactory.  It  is  difficult 
to  believe  that  the  effect  of  moderate  heat  is  to  increase  the  com- 
bining affinity  of  the  agglutinin,  whereas  greater  heat  destroys  it 
entirely,  and  it  is  altogether  different  to  what  is  observed  in  the 

21 — 2 


324  EXPLANATION    OF    SPECIFIC    INHIBITION 

case  of  toxin,  which  is  exactly  equal  to  toxoid  in  this  respect,  and 
Dreyer  and  Jex-Blake  bring  forward  very  strong  evidence  against 
this  view.  Some  .of  the  more  important  of  their  facts  may  be 
summarized  thus  :  If  it  were  true,  the  more  the  serum  is  weakened 
by  heat  (and  the  greater  the  production  of  agglutinoid)  the  greater 
should  be  the  zone  of  inhibition,  and  vice  versa.  This  they  found 
not  to  be  the  case,  for  a  serum  that  had  had  its  agglutinating  power 
very  largely  destroyed  by  heat  might  show  a  very  small  zone  of 
inhibition,  whilst  another  which  had  been  hardly  injured  might 
have  a  very  large  one.  Further,  a  serum  might  show  a  zone  with 
an  emulsion  of  bacteria  in  saline  solution,  but  not  in  broth. 

The  alternative  explanation  of  all  these  phenomena  is  based  on 
facts  observed  in  the  mutual  precipitation  of  colloids,  for  here 
again  exactly  similar  zones  of  inhibition  are  seen.  For  example, 
as  Neisser  and  Friedemann  have  shown,  a  suspension  of  particles 
of  mastic  in  water  (made  by  dropping  an  alcoholic  solution  into 
water)  takes  on  a  negative  charge,  and  can  be  precipitated  by 
positive  colloids  or  by  positive  ions.  Thus,  if  ferric  chloride  be 
added  precipitation  occurs,  and  if  the  dose  be  increased  it  gradually 
becomes  more  and  more  rapid  and  abundant  until  a  certain  amount 
of  ferric  chloride  is  present,  after  which  it  becomes  less  and  less 
until  no  reaction  takes  place  at  all.  And  similar  facts  have  been 
observed  in  numerous  other  cases  of  interaction  of  colloids.  Their 
explanation  is  somewhat  as  follows  :  When  the  two  colloids  are 
present  in  such  an  amount  that  their  electrical  charges  mutually 
annul  one  another,  and  they  are  therefore  formed  into  aggregates 
which  tend  to  run  together,  the  addition  of  fresh  colloid  bearing 
an  electric  charge  disturbs  the  conditions,  and  may,  by  electrifying 
the  masses  already  formed,  cause  mutual  repulsion,  and,  if  suf- 
ficient amount  of  the  second  colloid  be  added,  a  re-solution  of  all 
.the  masses.  Hence  solutions,  say,  of  an  electro-negative  colloid 
may  be  dependent  on  (a)  the  presence  of  mutually  repellent  mole- 
cules or  aggregates  of  molecules  in  an  inert  fluid,  or  (b)  on  the 
presence  of  these  molecules  in  a  fluid  also  containing  a  large 
number  of  molecules  of  opposite  sign,  whereas  in  the  intermediate 
mixtures  the  conditions  for  precipitation  are  present.  Here  the 
precipitate  is  soluble  in  excess  of  both  substances,  just  as  pre- 
cipitum  is  soluble  in  excess  either  of  precipitin  or  of  its  antigen. 

This  theory  of  the  action  of  agglutinins  has  been  investigated 
and  strongly  supported  by  Biltz,  Neisser  and  Friedemann,  Pauli, 
and  others,  and  Dreyer  and  Jex-Blake  have  in  particular  given 


COLLOIDAL   THEORY   OF   ANTIBODIES  325 

strong  evidence  in  its  favour.  For  example,  they  find  definite 
zones  of  inhibition  in  the  precipitation  of  B.  coli  by  orthophosphoric 
acid,  a  substance  in  which  the  existence  of  agglutinoids  is  out  of 
the  question.  Thus,  in  a  series  of  tubes  each  containing  1-5  c.c. 
of  an  emulsion  of  this  organism  in  normal  saline  solution,  agglu- 
tination occurred  in  the  tubes  to  which  amounts  from  118  centi- 
grammes down  to  4  centigrammes  were  added,  and  also  in  those 
containing  from  i-i  milligrammes  to  o-oi  milligramme,  but  there 
was  none  in  which  amounts  from  4  centigrammes  to  i'i  milli- 
grammes were  added.  Further,  there  is  a  close  analogy  between 
this  phenomenon  and  the  precipitation  of  gum  mastic  by  ferric 
chloride,  for  in  each  case  the  extent  of  the  zone  of  inhibition 
diminishes  as  the  emulsions  of  the  substance  precipitated  are  made 
stronger. 

The  facts  at  our  disposal  are  not  yet  sufficient  to  enable  the 
phenomena  of  agglutination  of  bacteria  to  be  fully  explained,  but 
the  process  may  take  place  somewhat  as  follows  :  Normal  bacteria 
pass  toward  the  anode  when  an  electric  current  is  passed  through 
the  fluid,  and  are  therefore  to  be  regarded  as  particles  carrying  a 
negative  charge.  The  addition  of  agglutinin  removes  this  charge, 
and  the  particles  become  electrically  inert.  This  is  shown  by  the 
fact  that  when  placed  in  an  electric  current  they  do  not  move  in 
either  direction.  If,  however,  very  large  amounts  of  agglutinin 
are  added,  the  point  of  neutrality  may  be  reached  and  passed,  and 
the  bacteria  acquire  a  positive  charge,  again  becoming  mutually 
repellent.  Remarkable  facts  which  may  have  some  bearing  on 
this  subject  have  been  brought  forward  by  Neisser  and  Friede- 
mann.  Emulsions  of  mastic  are,  as  we  have  seen,  precipitated  by 
ferric  hydroxide,  and  it  has  been  shown  that  the  addition  of  a 
small  amount  of  organic  colloid,  whether  positive  or  negative  or 
inert  like  gelatin,  protects  these  particles,  so  that  they  are  no  longer 
agglutinated  by  colloids  or  electrolytes  of  opposite  sign.  A  pheno- 
menon probably  similar  is  seen  when  salts  which  form  a  colloidal 
precipitate  are  mixed  in  a  solution  of  gelatin,  when  the  mixture 
remains  transparent,  the  colloidal  particles  being  prevented  from 
undergoing  flocculation.  Silver  chromate,  formed  by  the  inter- 
action of  potassium  chromate  and  silver  nitrate,  may  be  obtained 
in  a  transparent  and  stable  form  by  this  process.  Particles  thus 
protected  are  not  carried  in  either  direction  by  an  electric  current. 
Neisser  and  Friedemann  have  shown  that  mastic  emulsions  are 
"  protected  "  by  the  addition  of  a  little  serum,  leech  extract,  or 


326  COLLOIDS   AND   HAEMOLYSIS 

extract  of  typhoid  bacilli,  and  they  think  that  normal  bacteria 
may  be  compared  with  these  particles  surrounded  by  a  defensive 
envelope  which  prevents  the  flocculating  action  of  substances  of 
opposite  sign.  The  action  of  agglutinin  they  believe  to  consist  in 
the  removal  of  this  layer,  so  that  then  the  ions  of  opposite  sign 
can  unite  with  the  bacteria  and  bring  about  their  agglutination. 
This  explanation  would  explain  clearly  the  role  of  salts  in  agglu- 
tination, and  is  supported  by  Kirstein's  observation  that  typhoid 
bacilli  cultivated  in  medium  free  from  albuminoid  material  is 
clumped  by  salt  without  the  addition  of  serum.  The  theory,  how- 
ever, appears  at  present  not  to  account  for  the  facts  already  cited 
with  regard  to  the  transport  of  normal  and  charged  bacteria  in  an 
electric  current. 

Some  remarkable  analogies  have  been  brought  forward  by 
Landsteiner  and  Jagic  between  the  process  of  haemolysis  by 
simple  colloids  and  by  specific  antibodies.  Thus  a  solution  of 
colloidal  silicic  acid  acts  in  a  manner  closely  recalling  a  specific 
haemolysin.  It  clumps  and  dissolves  the  red  corpuscles  of  rabbits, 
and  agglutinates  their  spermatozoa,  but  has  no  action  on  typhoid 
bacilli ;  it  thus  shows  signs  of  selective  action,  though  not  of  true 
specificity.  Its  action  is  manifested  in  extremely  small  doses  ;  it 
is  rendered  inert  by  heat,  and  gradually  falls  off  even  at  the  room 
temperature.  Further,  phenomena  are  observable  which  strongly 
recall  the  action  of  complements  and  of  lecithin  in  reactivating 
heated  serum  or  in  dissolving  sensitized  corpuscles,  since  red 
corpuscles  which  have  been  agglutinated  by  colloidal  silicic  acid 
are  dissolved  by  traces  of  lecithin  or  of  fresh  serum,  but  not  by 
serum  which  has  been  heated  to  60°  C. ;  but  if  the  silicic  acid 
is  present  in  excess,  no  haemolysis  occurs. 

A  difficulty  arises  from  the  fact  that  emulsions  of  red  corpuscles 
are  agglutinated  by  both  positive  and  negative  colloids  (ferric 
hydroxide,  ferrocyanide  of  copper).  Girard-Mangin  and  Henri 
have  given  an  explanation  of  this,  which  is  briefly  as  follows  : 
When  a  red  corpuscle  is  suspended  diffusion  of  salts,  especially 
of  sulphates  of  calcium  and  magnesium,  takes  place  from  their 
surface,  and  these  facilitate  the  precipitation  of  positive  and 
negative  colloids  respectively,  so  that  the  corpuscles  come  to  be 
surrounded  by  a  layer  of  precipitated  colloid  material.  It  is  this 
zone  of  precipitated  material  which  actually  determines  the  agglu- 
tination of  the  corpuscles.  Thus  these  authors  consider  three 
substances  as  taking  part  in  the  process :  (a)  the  corpuscles ; 


COLLOIDAL   THEORY   OF   ANTIBODIES  327 

(b)  the  salts  which  gradually  diffuse  therefrom  ;  and  (c)  the 
colloid  or  agglutinin.  They  brought  forward  strong  evidence 
in  favour  of  this  theory  by  showing  that  agglutination  is  less 
powerful  when  the  corpuscles  are  suspended  in  normal  saline 
solution  (the  salts  of  which  inhibit  the  exosmosis  of  the  salts 
in  the  corpuscles)  than  when  they  are  in  isotonic  sugar  solu- 
tion (in  which  exosmosis  is  unchecked).  Further  deductions  from 
their  theory  (for  which  the  original  articles  must  be  consulted) 
were  verified  experimentally. 

The  analogies  between  colloid  adsorption  and  the  interactions 
of  toxin  and  antitoxin  have  been  studied  by  Biltz,  Pauli,  and 
others,  and  very  remarkable  facts  adduced.     For  instance,  there 
appear  to  be  phenomena  indicating  that  the  action  of  some  anti- 
toxins at  least  is  reversible  when  a  large  amount  is  present.   Thus, 
according  to  Jacoby,  the  action  of  crotin  (a  phytotoxin  of  simple 
nature   and   closely  analogous  with   the   bacterial   exotoxins)   is 
increased  by  the  addition  of  minute  amounts  of  antitoxin,  large 
quantities  of  which  neutralize  its  activity.     Phenomena  somewhat 
similar  are  seen  with  the  true  toxins — for  example,  Danysz  (cor- 
roborated by  von  Dungern  and  Sachs)  has  proved  that  diphtheria 
antitoxin  will  neutralize  more  toxin  when  added  at  once  than  when 
added  in  successive  portions.     Thus  if  the  amount  of  toxin  which 
is  just  neutralized  by  a  certain  amount  of  antitoxin  be  divided  into 
two  parts,  and  added  to  the  antitoxin  at  an  interval  of  twenty-four 
hours,  the  whole  may  be  toxic,  although  the  amounts  of  toxin  and 
of  antitoxin  present  are  exactly  the  same  in  the  two  cases.     Both 
these  effects  can  be  explained  on  Ehrlich's  pluralistic  conception. 
Thus  Jacobi's  results  may  be  explained  on  the  assumption  that 
small   amounts   of   anticrotin   combine   with   the   non-poisonous 
prototoxoid,  and  that  this  renders  the  mixture  more  toxic,  because 
this  prototoxoid  would  otherwise  seize  on  the  receptors  of   the 
sensitive  cells,  to  the  exclusion  of  the  active  toxin.   This,  however, 
is  very  difficult  to  believe.     Taking  the  action  of  crotin  in  pro- 
ducing haemolysis  as  a  test,  it  would  appear  that  the  neutralization 
of  some  of  the  toxin,  even  if  inert,  would  render  the  substance 
less  haemolytic  ;  this  appears  to  follow  from  Bordet's  proof  that 
red  corpuscles  can  take  up  much  more  than  their  haemolyzing  dose 
of   an  antibody.     The  Danysz  effect  is  also  explicable  on   the 
theory  of  the  presence  of  a  non-toxic  epitoxonoid  of  no  toxicity 
and  of  feeble  combining  affinity.     This  slowly  forms  a  firm  com- 
bination with  the  antitoxin,  and  renders  it  useless  for  neutralizing 


328         ANALOGIES  WITH  DANYSZ'  EFFECT 

more  toxin.  Explanations  are  also  available  on  the  Arrhenius- 
Madsen  explanation,  regarding  the  combinations  as  examples  of 
mass  reaction. 

Regarding  reactions  such  as  these  as  interactions  of  colloids, 
we  find  them  paralleled  in  simple  reactions.  Thus  in  some  cases 
the  addition  to  a  colloidal  solution  of  a  small  amount  of  a  second 
colloid  of  opposite  sign  may  render  the  solution  more  stable,  and 
protect  it  from  precipitation  by  an  excess  of  the  second  substance. 
The  partially  neutralized  aggregates  appear  to  possess  more  re- 
pulsive power  than  those  carrying  their  full  electric  charge. 
Again,  the  effect  of  an  addition  of  one  colloid  to  another  of 
opposite  sign  is  often  brought  about  slowly,  and  the  material  only 
gradually  attains  its  permanent  form.  It  is  probably  this  fact 
that  renders  solutions  of  antitoxin  so  unstable.  Interreactions  take 
place  between  the  colloids  themselves  and  the  electrolytes  in  the 
serum,  aggregates  are  formed,  and  the  antitoxic  potency  falls  off— 
rapidly  at  first  and  subsequently  more  slowly.  It  is  a  matter  of 
great  difficulty  to  prepare  stable  solutions  of  proteid  materials. 
As  we  have  already  pointed  out,  the  amount  of  colloid  necessary 
to  precipitate  a  constant  amount  of  another  of  opposite  sign  is 
reduced  to  a  minimum  if  the  addition  be  made  at  once,  and 
rendered  much  greater  if  it  is  made  slowly  in  small  amounts  with 
an  interval  between  each.  This  is  closely  analogous  with  the 
Danysz  effect.  And  this  must  also  be  considered  in  its  bearing 
on  Ehrlich's  method  of  attempting  to  neutralize  a  certain  dose  of 
toxin  by  successive  addition  of  small  amounts  of  antitoxin,  the 
method  by  which  the  whole  of  the  elaborate  pluralistic  conception 
of  the  structure  of  toxins  has  been  built  up.  According  to  the 
colloidal  theory,  this  is  explicable  on  the  assumption  that  tran- 
sitional compounds  of  toxin  and  antitoxin  of  very  diverse  nature, 
not  necessarily  dependent  on  the  proportions  of  the  two  substances, 
are  formed.  This  is  practically  the  view  put  forward  by  Bordet 
from  a  consideration  of  the  reactions  of  the  antibodies,  and  especially 
alexin  and  anti-alexin,  and  supported  by  the  workers  who  have 
examined  the  question  from  the  standpoint  of  colloidal  reactions. 

The  difference  between  the  L0  and  L+  dose,  which  is  so 
important  a  feature  in  Ehrlich's  work,  also  finds  an  analogy  in 
the  reactions  of  simple  colloidal  substances.  Thus  ferric  hydroxide 
neutralizes  arsenious  acid  (whence  its  use  as  an  antidote  in  acute 
arsenical  poisoning),  and  it  was  found  by  Biltz  that  if  one  lethal 
dose  of  the  latter  substance  was  added  to  a  neutral  mixture  of  the 


COLLOIDAL   THEORY    OF   ANTIBODIES  32Q 

two,  this  mixture  did  not  regain  toxicity.  Several  lethal  doses 
must  first  be  added,  just  as  it  is  necessary  to  add  several  lethal 
doses  of  diphtheria  toxin  to  the  L0  dose. 

A  difficulty  arises  from  the  fact  that  both  toxin  and  antitoxin 
pass  in  the  same  direction  in  an  electric  current.  These  experi- 
ments are  naturally  difficult  to  carry  out,  since  the  effect  of 
electrolysis  must  be  eliminated.  When  this  is  done,  according  to 
Field  and  Teague,  both  substances  travel  towards  the  cathode,  and 
this  whether  the  solution  is  acid  or  alkaline.  But  we  must 
remember  that  when  toxin  and  antitoxin  interact  they  always  do 
so  in  a  very  complex  fluid,  containing  many  other  substances, 
both  colloids  and  electrolytes,  and  until  we  can  determine  the 
electric  charge  of  these  substances  in  a  pure  form,  these  experi- 
ments can  hardly  be  considered  to  outweigh  the  remarkable 
analogies  described  above.  It  is  highly  probable,  judging  from 
analogy  with  the  other  antibodies,  that  free  ions  are  essential  to 
the  neutralization  of  toxin  by  antitoxin,  and  the  researches  of 
Girard-Mangin  and  Henri  show  us  how  complex  the  conditions 
may  become. 

We  have  seen  that  it  is  important  to  know  whether  the 
compounds  of  antibody  and  antigen  are  dissociable,  and  that  the 
latter  view  explains  some  difficult  phenomena  seen  in  immunity. 
On  Ehrlich's  theory  the  combination  between  the  two  is  a  strong 
one,  and  once  it  has  been  allowed  to  take  place,  the  two  substances 
are  not  dissociated  into  their  components,  whereas  on  Arrhenius's 
and  Madsen's  theory  the  combination  is  an  unstable  one,  and  dis- 
sociation constantly  occurs.  The  experimental  proof  in  favour  of 
the  occurrence  of  this  phenomenon  appears  on  the  whole  satisfac- 
tory. Red  corpuscles  saturated  with  immune  body  mixed  with 
normal  corpuscles  will  part  with  some  of  their  antibody,  so  that 
the  latter  become  sensitized.  A  mixture  of  tetanus  toxin  and 
antitoxin,  which  causes  no  symptoms  when  injected  into  the  leg  of 
a  guinea-pig,  causes  tetanus  when  adrenalin  has  previously  been 
injected  locally  (explicable  on  the  fact  that  toxin  is  absorbed  by, 
the  nerves  and  antitoxin  by  the  vessels,  which  in  the  second  case 
are  constricted),  and  other  examples  might  be  quoted.  It  appears, 
however,  that  this  irreversibility  only  occurs  if  the  two  substances 
are  not  kept  in  contact  for  a  sufficiently  long  time,  as  was  pointed 
out  by  Martin  and  Cherry  in  their  nitration  experiments  very 
early  in  the  history  of  the  chemistry  of  the  action  of  antitoxin. 
Thus  in  the  latter  example,  if  the  tetanus  toxin  and  antitoxin  be 

I 


330  COLLOIDAL   CHEMISTRY 

kept  in  contact  for  two  hours,  no  tetanus  is  produced,  in  spite  of 
the  local  action  of  the  adrenalin.  This  appears  to  harmonize  best 
with  the  colloidal  theory.  The  interactions  of  colloids  of  opposite 
sign  and  the  precipitation  of  colloids  by  electrolytes  may  be 
reversible  if  the  conditions  are  changed  early,  but  the  compounds 
formed  gradually  become  more  and  more  stable.  But  if  the  con- 
tinuation were  that  of  two  substances  of  strong  chemical  affinity 
we  should  expect  it  to  be  stable  from  the  first,  and  if  it  followed 
the  laws  of  mass  reaction  it  would  be  reversible,  no  matter  how 
long  it  had  stood. 

There  appears  to  be  no  crucial  experiment  by  which  the  truth 
of  these  three  theories  can  be  tested.  The  arguments  brought 
forward  by  the  upholders  of  each  involve  the  study  of  the  exceed- 
ingly complicated  laws  connected  with  the  true  relations  and 
completeness  of  the  reactions,  and  involve  advanced  mathematical 
consideration.  It  is,  however,  doubtful  whether  the  processes  are 
measurable  with  the  accuracy  required  in  the  investigation  of 
these  complicated  formulae,  and  in  some  cases  very  slight  altera- 
tion of  the  observed  results  would  render  the  facts  explicable  on 
a  theory  other  than  that  in  support  of  which  it  was  adduced.  At 
the  present  time  the  balance  of  the  evidence  appears  to  be  decidedly 
in  favour  of  the  colloid  hypothesis. 


CHAPTER  XIII 
ON  IMMUNITY  TO  BACTERIA 

WE  are  now  in  a  position  to  discuss  the  mechanism  by  which  a 
bacterial  infection  is  combated.  It  must  be  pointed  out  in  the 
first  place,  and  kept  in  mind  throughout,  that  different  microbes 
are  dealt  with  in  different  ways.  Some,  such  as  many  of  the 
protozoan  blood  parasites,  may  be  tolerated,  since  the  tissues  of 
their  host  are  immune  to  the  action  of  their  toxins ;  others  are 
removed  by  phagocytosis,  after  preparation  by  opsonins,  or  perhaps 
by  alexins,  amboceptors,  or  other  substances,  or  possibly  without 
previous  treatment ;  and  yet  others  may  be  dissolved  by  the  process 
discussed  under  the  heading  of  Bacteriolysis.  Each  of  these 
processes  gives  rise  to  a  different  form  of  immunity,  which  we 
may  call — (i)  immunitas  non  sterilisans,  (2)  phagocytic  immunity, 
and  (3)  bacteriolytic  immunity.  And  the  latter  two  processes 
(which  are  the  only  ones  of  practical  importance)  rarely,  if  ever, 
exist  in  a  pure  state ;  almost  all  invading  bacteria  are,  or  may  be, 
dealt  with  by  a  combination  of  the  two. 

We  must  also  notice  that  the  defensive  processes  will  be  greatly 
modified  by  the  nature  of  the  region  in  which  the  combat  between 
the  invaders  and  the  defensive  mechanism  goes  on.  There  are 
three  main  cases  :  (i)  the  circulating  blood,  (2)  the  tissues,  and 
(3)  regions,  such  as  the  peritoneum,  combining^some  of  the 
characters  of  the  other  two.  We  will  take  them  Jh  this  order. 

In  dealing  with  the  processes  that  take  place  when  bacteria  gain 
access  to  the  circulating  blood,  our  main  difficulty  at  first  sight  is 
not  to  explain  the  existence  of  immunity,  but  the  reason  why 
infection  ever  occurs  ;  for  the  defensive  mechanism  seems  more 
than  adequate  to  combat  any  dose  of  bacteria  which  is  likely  to 
gain  access  to  the  blood  under  natural  conditions.  The  processes 
by  which  the  bacteria  are  removed  are  phagocytosis  and  bacterio- 
lysis, or  the  two  combined,  and  in  either  case  the  defence  would 
appear  to  be  more  than  adequate.  Thus,  in  an  experiment  for  the 

331 


332  DIFFICULTIES   OF   THE    PROBLEM 

determination  of  the  opsonic  index  in  which  thick  emulsions  of 
bacteria  are  used  the  leucocytes  are  often  found  absolutely  packed 
with  streptococci  or  tubercle  bacilli  after  a  short  incubation  ;  if 
on  the  average  100  cocci  are  taken  up,  calculation  will  show  that 
the  whole  volume  of  blood  might  phagocyte  2,000,000,000,000  in 
a  few  minutes.  Tubercle  bacilli  are  perhaps  hardly  so  easily 
taken  up,  but  it  is  difficult  to  see  how  the  comparatively  moderate 
number  of  these  bacteria  which  gain  access  to  the  blood  after  the 
rupture  of  a  small  caseous  gland  into  a  vein  can  escape  immediate 
phagocytosis :  yet  we  have  no  reason  to  believe  that  this  accident 
often  occurs  without  causing  general  tuberculosis.  Perhaps  a 
more  convincing  example  is  given  by  determinations  of  the  opsonic 
index  in  septicaemia,  where  the  bacteria— e.g.,  streptococci — are 
known  to  be  circulating  in  the  blood.  Here  the  opsonic  index  is 
usually  low  (0-5  or  less),  but  even  then  it  is  sufficient  to  enable  the 
leucocytes  to  take  up  an  enormously  greater  number  of  bacteria 
than  we  can  discover  in  the  blood. 

Similar  phenomena  occur  in  the  case  of  bacteria  which  are  com- 
bated largely  by  means  of  bacteriolysins.  Thus,  normal  blood 
serum  is  markedly  bactericidal  to  typhoid  bacilli,  yet  infection 
occurs,  and  many  observers  have  noticed  relapses  when  the 
bacteriolytic  power  of  the  blood  is  extremely  high.  In  these 
cases,  therefore,  the  bacteria  can  gain  access  to  the  blood  (for 
typhoid  fever  is  always  in  part  a  septicaemia)  in  spite  of  barriers 
which  would  appear  unsurmountable.  And  we  have  already  had 
occasion  to  mention  that  anthrax  may  occur  in  animals  the  blood 
serum  of  which  is  powerfully  bactericidal ;  the  animals,  indeed, 
may  be  extremely  susceptible. 

Before  attempting  to  explain  the  apparent  discrepancy,  it  must 
be  pointed  out  that,  as  a  matter  of  fact,  septicaemia  is  a  rare 
disease  in  proportion  to  the  opportunities  for  its  occurrence,  and 
that  the  continued  presence  of  bacteria  in  the  blood  without  a  local 
lesion  from  which  they  are  constantly  discharged  is  extremely 
uncommon.  Thus  in  diphtheria,  tetanus,  the  staphylomycoses, 
etc.,  in  which  we  should  imagine  that  there  is  every  chance  for 
bacteriaemia  to  occur,  it  is  practically  unknown.  It  is  common  in 
diseases  like  ulcerative  endocarditis,  the  early  stages  of  typhoid 
fever,  and  in  pneumonia,  in  which  we  have  reason  to  believe  that 
there  is  a  constant  shower  of  organisms  discharged  from  the  local 
lesion  direct  into  the  blood.  Here,  however,  we  can  feel  fairly 
certain  that  the  bacteria  which  we  find  in  the  blood  are  only  there 


ON    IMMUNITY    TO    BACTERIA  333 

temporarily,  and  are  rapidly  destroyed  :  the  reasons  being,  firstly, 
that  they  are  never  present  in  large  numbers,  as  they  would  be  if 
they  lived  and  multiplied  in  the  circulation,  and,  secondly,  that 
they  do  not  as  a  rule  form  secondary  lesions,  as  we  might  expect 
them  to  do,  when  deposited  in  the  tissues.  We  may  fairly  assume, 
therefore,  that  in  a  disease  like  typhoid  fever  bacteria  are  constantly 
passing  from  the  lesions  into  the  blood,  but  are  rapidly  destroyed, 
so  that  the  defensive  mechanism  of  the  fluid  is  sufficient  to  prevent 
the  occurrence  of  a  true  septicaemia,  in  which  bacterial  growth 
continues  in  the  blood.  The  same  holds  in  pneumonia  and  in 
ulcerative  endocarditis  when  not  accompanied  by  secondary 
infective  lesions,  and  probably  most  other  diseases,  and  does  not 
militate  against  the  view  that  the  blood  is  a  sufficient  defence  in 
the  majority  of  the  cases  in  which  the  infective  material  gains 
access  to  the  interior  of  the  vessels.  Excluding  these  and  similar 
examples,  septicaemia  is  a  rare  disease,  and  is  commoner  in  the 
case  of  the  protozoal  than  in  bacterial  infections. 

As  regards  the  failure  of  the  process  of  phagocytosis,  which 
must  occur  when  an  organism  which  is  combated  by  phagocytosis 
does  gain  a  foothold  in  the  blood,  we  may  point  out  that  we  have 
yet  but  little  knowledge  of  the  exact  state  of  the  bacteriotropic 
substances  in  the  living  plasma.  Wright,  it  is  true,  has  shown 
that  citrated  plasma  contains  the  same  amount  of  opsonin  as 
serum ;  but  the  two  fluids  are  not  absolutely  identical,  and  the  fact 
that  opsonin  increases  in  amount  in  the  successive  fractions  of 
serum  squeezed  out  from  a  clot  leads  us  to  believe  it  probable 
that  it  does  not  exist  as  such  in  the  plasma ;  and  if,  as  we  have 
shown  is  likely,  the  naturally  existent  thermolabile  opsonin  is  the 
same  as  alexin,  it  is,  on  the  whole,  probable  that  there  is  none  in 
the  living  blood. 

Briscoe's  experiments  reproduce  the  natural  conditions  more 
closely.  He  injected  emulsion  of  bacteria  into  the  ventricle  of  a 
heart  after  excision  and  clamping  of  the  auriculo-ventricular 
groove.  After  a  time  some  of  the  blood  was  removed  and 
examined,  and  practically  no  phagocytosis  was  found  to  have 
taken  place.  But  many  bacteria  were  taken  up  when  the  same 
animal's  leucocytes  and  serum  and  the  emulsion  of  bacteria  were 
incubated  outside  the  body  in  the  ordinary  way.  Further,  in 
some  cases  a  little  clotting  occurred  in  the  heart,  and  in  these  it 
was  found  that  the  leucocytes  which  were  included  within  the 
clot  took  up  many  more  bacilli  than  those  which  remained  free. 


334          FUNCTION    OF   THE    SPLEEN    IN    PHAGOCYTOSIS 

These  results  argue  strongly  in  favour  of  the  fact  that  opsonin 
does  not  occur  in  the  circulating  plasma  as  such,  and  is  only  set 
free  during  the  process  of  coagulation  or  of  phagolysis.  This 
conclusion  was  corroborated  by  the  fact  that  opsonized  bacteria 
were  taken  up  freely,  showing  that  the  leucocytes  were  not  at 
fault, 

We  can  get  no  information  on  this  point  from  an  examination 
of  the  blood  in  these  infections,  since  a  small  number  of  non- 
virulent  bacteria  will  be  digested  and  completely  disappear  in  a 
few  minutes  if  taken  up  by  a  leucocyte  ;  but  as  a  matter  of  fact,  it 
is  very  uncommon  to  find  ingested  bacteria  in  the  leucocytes  of 
the  circulating  blood  in  natural  diseases. 

It  might  appear  that  these  explanations  go  too  far,  and  would 
lead  us  to  the  conclusion  that  phagocytosis  is  without  value  as  a 
defensive  process  when  bacteria  reach  the  blood-stream.  This  is 
not  the  case,  and  it  is  probable  that  the  great  factor  to  consider  is 
the  spleen.  This  acts  as  a  sort  of  filter,  and  in  its  pulp  the  flow 
of  blood  is  slow,  so  that  there  is  abundant  opportunity  for  phago- 
cytosis to  occur.  The  same  is  true,  though  in  a  lesser  degree,  of 
all  regions  of  the  body  in  which  the  circulation  is  tardy.1  Hence, 
when  bacteria  gain  access  to  the  blood  they  will  always  be  found 
in  considerable  numbers  in  this  organ,  which  Kanthack  called 
years  ago  "  the  graveyard  of  the  bacteria."  This  is  especially  the 
case  in  typhoid  fever,  and  the  method  formerly  in  use  in  the 
diagnosis  of  the  disease,  by  making  cultures  from  traces  of  spleen 
pulp  removed  by  puncture,  almost  invariably  gave  positive  results. 
Films  or  sections  of  the  spleen  pulp  will  often  show  bacteria 
ingested  by  the  leucocytes  in  many  diseases,  even  although  none 
can  be  detected  in  the  blood.  The  role  of  the  spleen  in  immunity 
is  probably  a  complex  one,  and  this  may  explain  the  divergent 
views  held  as  to  its  importance  in  this  respect.  Thus  it  was 
found  long  ago  by  Tizzoni  and  Cattani  that  it  was  impossible  to 
immunize  animals  to  tetanus  after  splenectomy ;  but  this  was  not 
corroborated  by  other  observers,  who  found  the  animals  equally 
immunizable  after  the  results  of  the  operation  had  completely 
passed  away.  But  in  this  connection  we  must  remember  that  the 
splenic  tissue  is  very  rapidly  regenerated,  and  that  in  all  prob- 
ability the  haemolymph  glands  and  other  structures  take  on  its 
functions  vicariously  after  its  ablation.  ^ 

1  Leucocytes  which  have  taken  up  bacteria  whilst  in  the  circulation 
probably  accumulate  in  the  lungs  also. 


ON    IMMUNITY    TO    BACTERIA  335 

The  role  of  the  spleen  in  facilitating  phagocytosis  is  well  seen 
in  the  spirilloses.  Metchnikoff  showed  many  years  ago  that  after 
death  from  relapsing  fever  the  cells  of  this  organ  are  packed  with 
spirilla,  such  cells  being  rarely,  if  ever,  seen  in  the  circulating 
blood.  When  monkeys  are  inoculated  with  blood  containing  the 
parasite,  a  febrile  attack  occurs,  but  recovery  soon  takes  place. 
During  the  disease  free  spirilla  occur  in  the  blood,  but  they  soon 
disappear ;  but  if  the  animals  are  killed  after  this,  the  organisms 
may  be  found  within  the  splenic  leucocytes.  Further,  Souda- 
kewitch  showed  that  recovery  often  failed  to  occur  in  animals 
inoculated  after  splenectomy,  and  in  this  case  the  spirillum 
appeared  in  large  and  increasing  numbers  in  the  blood,  and  no 
phagocytosis  was  observed.  Very  similar  facts  have  been 
observed  by  Levaditi  and  Manouelian  in  tick-fever.  During  the 
height  of  the  disease  the  spirillum  occurs  in  the  blood,  and  is 
exclusively  extracellular.  After  the  crisis  it  is  found  only  in  the 
phagocytes  of  the  spleen  and  liver  (Kupffer's  cells). 

Besides  the  spleen,  the  bone-marrow  is  also  a  region  where 
abundant  phagocytosis  occurs,  and  the  functions  of  the  two  tissues, 
like  their  structure,  are  similar.  In  each  case  there  is  a  slow  flow^ 
of  blood  in  the  pulp,  close  proximity  of  the  blood  to  the  tissue 
cells  of  peculiar  type,  and  an  abundant  supply  of  leucocytes. 

The  function  of  these  organs  in  phagocytosis  is  probably  two- 
fold. It  provides  a  region  where  the  blood-stream  is  comparatively 
tranquil,  so  that  there  is  no  mechanical  obstacle  to  the  process : 
this  is  probably  comparatively  unimportant,  since  it  can  and  does 
take  place  under  certain  circumstances  in  the  full  torrent  of  the 
circulation.  And,  secondly,  if,  as  seems  probable,  thermolabile 
opsonin  is  really  complement,  and  if  complement  is  formed  by  the 
leucocytes,  it  is  readily  conceivable  that  it  may  be  present  in  the 
leucocytic  organs  when  only  present  in  small  amount  in  the  rest 
of  the  blood.  It  may  actually  be  called  into  existence  by  the  action 
of  the  toxin  on  the  spleen  cells  or  leucocytes,  and  be  immediately 
absorbed  by  the  bacteria,  so  that  it  never  occurs  in  appreciable 
amount  in  the  plasma. 

This  action  will  not  come  into  play  in  the  case  of  the  bacterio- 
tropic  substances  which  are  true  antibodies,  for  we  have  every 
reason  to  believe  that  these  are  present  in  the  plasma  as  such,  and 
are  not  set  free  in  the  process  of  clotting ;  yet  even  here  we  have 
seen  some  evidence  which  suggests  that  they,  in  common  with 
other  true  antibodies,  are  formed  inlymphoid  tissue  or  from  lymphoid 


336  FUNCTIONS    OF    OTHER    ORGANS 

cells,  or,  as  some  recent  research  seems  to  prove,  from  the  endo- 
thelium. 

The  liver  is  also  an  important  region  with  regard  to  the  deposi- 
tion of  bacteria,  and  their  subsequent  destruction  by  phagocytosis 
or  other  means,  but  in  this  case  the  main  cells  concerned  are 
Kupffer's  cells.  We  must  regard  these  two  organs  and  the  lungs 
as  being  intercalated  in  the  circulation  with  the  object,  amongst 
others,  of  arresting  bacteria  and  other  foreign  particles  which  gain 
access  to  the  circulation,  and  affording  a  region  in  which  phagocytosis 
can  take  place  at  leisure.  It  has  been  shown  experimentally  that 
animals  will  resist  a  much  larger  dose  of  bacteria  if  the  injection 
be  made  into  the  portal  vein  than  if  it  is  thrown  into  one  of  the 
peripheral  veins.  The  importance  of  these  organs  is  also  well 
shown  from  a  study  of  the  fate  of  inert  particles,  such  as  carbon 
or  carmine,  when  injected  into  the  circulation  or  peritoneum.  In 
either  case  they  make  their  way  with  great  rapidity  to  the  organs, 
especially  the  liver  and  spleen,  in  which  they  occur  in  large 
numbers  within  two  hours  of  the  injection.  They  are  also  found 
within  the  bone-marrow,  lymph  glands,  tonsils,  and  lungs,  and, 
whatever  the  region  in  which  they  occur,  are  mostly  contained 
within  the  leucocytes  or  tissue  phagocytes.  Here  also  intracellular 
absorption  may  take  place,  though  of  course  it  is  much  slower 
than  is  the  case  with  bacteria,  and  according  to  Siebel  the  leuco- 
cytes, with  their  load  of  unabsorbable  particles,  may  leave  the  body 
via  the  lung,  tonsils,  or  lymphoid  structures  of  the  small  intestine. 

The  lung  plays  a  part  of  great  importance,  but  one  not  fully 
understood,  in  the  process  of  phagocytosis  of  bacteria  which  gain 
access  to  the  circulation.  According  to  some  writers,  it  is  the  only 
internal  organ  in  which  phagocytosis  by  polynuclears  can  be 
demonstrated,  but  this  is  certainly  not  correct.  It  is  true,  how- 
ever, that  soon  after  the  injection  of  streptococci  (Tchistovitch)  or 
cholera  vibrios  (Levaditi)  into  a  rabbit,  polynuclears  containing 
these  organisms  may  be  found  in  large  numbers  in  the  wide 
capillaries  of  the  lung ;  this  is  probably  the  reason  for  the  sudden 
fall  in  the  number  of  the  leucocytes  in  the  circulating  blood  which 
occurs  soon  after  injection.  The  reason  for  this  collection  in  the 
lungs  is  very  far  from  clear :  the  region,  remote  as  it  is  from 
lymphoid  tissue,  would  appear  an  unfavourable  battle-ground 
against  the  infection ;  nor  are  the  later  stages  of  the  process  fully 
understood. 

The  question  of  the  importance  of  the  bacteriolysins  in  haemic 


ON    IMMUNITY   TO    BACTERIA  337 

infections,  already  alluded  to,  now  requires  further  mention.  It 
is  obvious  that  the  role  which  it  plays  varies  greatly  in  different 
diseases.  There  is,  for  instance,  no  evidence  for  believing  that 
the  blood  ever  develops  any  substance  which  is  bacteriolytic  for 
the  tubercle  bacillus.  It  is  true  that  the  researches  of  Wassermann 
and  his  coadjutors  show  (by  means  of  the  method  of  fixation  of 
complement)  that  an  antibody  may  occur  in  tuberculosis  or  in 
tuberculous  animals,  but  there  is  no  evidence  that  this  is  a 
bacteriolysin,  or,  if  so,  that  it  ever  occurs  in  amounts  sufficient  to 
bring  about  solution  of  tubercle  bacilli ;  in  fact,  it  appears  probable 
that  tubercle  may  run  its  whole  course  without  leading  to  the  pro- 
duction of  any  recognizable  antibody.  So,  too,  with  staphylococcic 
infections.  Here  it  is  possible  by  special  methods  to  produce  a 
serum  which  has  a  slight  bactericidal  effect,  but  normal  human 
serum  or  the  serum  of  a  patient  who  has  been  submitted  to  a 
course  of  antistaphylococcic  vaccination  is  quite  powerless  in  this 
respect.  It  is  obvious,  therefore,  that  neither  the  high  grade  of  natural 
immunity  to  staphylococci  of  normal  persons  nor  the  increased 
amount  present  in  active  immunity  is  due  to  any  bactericidal  or 
bacteriolytic  substances  occurring  in  the  blood.  It  is  quite  true 
that  in  old  specimens  of  staphylococci  pus-free  cocci  in  all  stages 
of  degeneration  can  be  found,  rendering  it  quite  obvious  that  a 
process  of  extracellular  death  and  destruction  of  bacteria  is  at 
work.  This  phenomenon  is  probably  to  be  attributed  mainly  to 
the  action  of  the  proteolytic  enzymes  formed  by  the  leucocytes  of 
the  pus.  These  are  substances  which  are  too  often  neglected  in  con- 
sideration of  immunity.  They  are,  of  course,  non-specific,  and  will  act 
on  any  dead  proteid,  and  on  some  living  organisms.  Their  action  may 
be  demonstrated  by  allowing  some  liquor  puris  from  an  old  abscess 
to  act  (in  presence  of  thymol)  on  some  staphylococci  which  have 
been  previously  boiled  to  prevent  autolysis.  Another  process  in 
which  these  degenerated  forms  of  cocci  may  be  produced  is  by 
spontaneous  autolysis  in  organisms  killed  by  lack  of  suitable 
nourishment,  or  atrepsy. 

In  sharp  distinction  from  tubercle  and  the  staphylomycoses,  we 
find  such  diseases  as  cholera  and  typhoid  fever,  in  which  bacterio- 
lysins  are  formed  in  large  amounts,  and  are  readily  demonstrable 
by  simple  methods.  It  is  in  these  and  a  few  other  diseases  that 
we  may  expect  the  role  of  these  substances  in  recovery  and 
immunity  to  be  most  marked.  We  find,  however,  that  their 
action  is  most  difficult  to  understand,  and  that  its  importance 

22 


ROLE    OF  THE    BACTERIOLYSINS    IN    RECOVERY 

appears  to  diminish  the  more  it  is  investigated.  In  general  terms, 
there  is  no  doubt  as  to  the  fact  that  the  presence  of  abundant 
bacteriolytic  substances  must  be  regarded  as  a  cause  of  immunity : 
thus,  animals  may  be  rendered  passively  immune,  e.g.,  to  intra- 
peritoneal  injections  of  typhoid  or  cholera  by  antecedent  or 
simultaneous  injections  of  powerful  bactericidal  serum.  But 
further  investigation  shows  that  even  in  this  phenomenon  there 
are  certain  features  which  must  lead  us  to  regard  the  process  of 
bacteriolysis  as  of  subordinate  importance,  or  even  as  harmful. 
In  the  first  place,  the  demonstration  of  the  fact  that  the  opsonic 
action  of  a  powerful  antityphoid  serum  (as  determined  by  the 
process  of  dilution  until  extinction  of  the  property  occurs)  is  pro- 
portionate to  its  bactericidal  action,  and  other  facts  already  noticed, 
have  led  us  to  the  belief  that  immune  body  or  amboceptor  can 
play  the  part  of  an  opsonin,  and,  further,  that  it  does  so  when 
present  in  amount  so  small  that  its  bacteriolytic  action  is  but  slight 
or  not  apparent.  It  is,  therefore,  in  the  highest  degree  probable 
that  the  passive  immunity  conferred  by  the  injection  of  a  bacterio- 
lytic serum  is  due  to  the  opsonizing  action  of  this  serum,  manifested 
even  when  diluted  with  the  juices  of  the  animal  into  which  it  is 
injected. 

In  the  second  place,  there  is  this  main  difference  between  the 
destruction  of  bacteria  within  the  leucocyte  after  phagocytosis 
has  occurred  and  the  extracellular  solution  by  immune  body  and 
alexin :  in  the  former  case  it  is  unusual  for  the  endotoxins  to  be 
set  at  liberty,  so  that  as  soon  as  a  bacterium  has  been  taken  up  by 
a  leucocyte,  we  believe  that,  as  a  rule,  its  capacity  for  harm  has 
been  removed.  It  undergoes  solution  within  the  protoplasm  of  the 
leucocyte,  which,  as  appears  from  the  studies  of  the  French  school, 
is  peculiarly  insusceptible  to  the  action  of  toxins,  is  destroyed  by 
the  digestive  juices,  or  by  other  means,  and  is  not,  at  least  in  the 
majority  of  cases,  set  free  to  injure  the  tissue  cells.  It  is  other- 
wise with  the  solution  of  bacteria  which  takes  place  from  the 
action  of  bacteriolytic  antibodies,  for  here  the  endotoxins  are  set 
free,  so  that  the  presence  of  these  antibodies,  though  undoubtedly 
developed  as  a  part  of  the  defensive  mechanism,  may  be  a  source 
of  added  danger  to  the  animal.  For  example,  if  an  intraperitoneal 
injection  of  a  large  dose  of  cholera  vibrios  be  made  into  the  peri- 
toneal cavities  of  two  animals,  and  in  one  some  anticholera  serum 
be  added,  it  is  often  found  that  this  animal  dies  very  rapidly  with 
symptoms  of  acute  intoxication,  whilst  the  other  survives  longer 


ON    IMMUNITY   TO    BACTERIA  339 

and  dies  of  septicaemia.  If  dead  vibrios  are  used  the  addition  of 
serum  may  bring  about  death,  whilst  an  animal  injected  with  the 
organisms  alone  may  recover  with  scarcely  a  symptom  of  intoxica- 
tion. Similar  phenomena,  though  less  definite,  often  follow  the 
use  of  antistreptococcic  serum  for  patients  suffering  from  strepto- 
coccic  disease :  the  injection  may  cause  a  rapid  rise  of  the 
temperature  and  exacerbation  of  the  symptoms.  Other  explana- 
tions are  possible,  but  it  is  highly  probable  that  this  is  due  to  the 
solution  of  some  of  the  cocci  and  liberation  of  their  endotoxin. 
It  is  possible  that  in  some  cases  this  might  be  a  source  of  danger, 
and  that  this  liberated  toxin  might  be  sufBcient  to  kill  the  patient ; 
but  there  is  no  evidence  that  this  actually  occurs,  and,  as  a  rule, 
the  phenomena  mentioned  may  be  taken  as  proof  that  the  serum 
is  a  suitable  one,  and  that  its  use  should  be  continued. 

We  must,  however,  be  careful  in  arguing  from  the  phenomena 
occurring  in  hypervaccinated  animals  to  those  suffering  from  a 
natural  infection.  There  can  be  no  analogy  between  the  train  of 
events  when  a  massive  dose  of  pathogenic  bacteria  is  suddenly 
injected  into  the  circulation  or  peritoneal  cavity  of  an  animal  in 
which  there  is  already  an  abundance  of  bacteriolytic  substances, 
and  those  which  follow  a  natural  infection.  Considering  the 
latter  case,  and  excluding  all  considerations  of  phagocytic  action, 
we  may  trace  the  sequence  of  events  in  the  case  of  a  disease  such 
as  typhoid  fever  somewhat  as  follows  :  In  the  first  place,  since 
normal  human  serum  is  bactericidal  to  typhoid  bacilli,  we  must 
regard  the  normal  resistance  against  the  disease  as  being  due,  in 
part  at  least,  to  the  presence  of  immune  body  in  the  serum, 
though  we  have  no  means  of  knowing  whether  its  action  is  mainly 
opsonic  or  mainly  bacteriolytic  when  occurring  in  the  plasma. 
When  infection  occurs,  the  amount  of  this  substance  must 
diminish,  since  it  will  combine  with  the  typhoid  bacilli  to  which 
it  gains  access,  and  may,  indeed,  be  sufBcient  to  kill  them  all,  and 
thus  prevent  the  development  of  the  disease.  Where,  however, 
the  organisms  gain  access  in  sufficient  numbers,  so  that  the  amount 
of  immune  body  or  of  alexin  is  insufficient  to  prevent  the  further 
growth  of  some  of  them,  those  that  escape  will  continue  to  grow, 
and  the  disease  will  gradually  increase  in  severity.  At  the  same 
time,  the  dissolved  products  of  the  organisms  which  succumb  will 
reach  the  seats  of  antibody  production  and  stimulate  a  fresh  pro- 
duction of  immune  body.  WTe  have  seen  reason  to  believe  that 
this  production  takes  place  mainly  in  the  lymphoid  tissue  and 

22 — 2 


340       SEQUENCE    OF    EVENTS    IN    GENERAL    INFECTIONS 

other  regions  rich  in  lymphocytes,  and  we  may  probably  corrobo- 
rate this  fact  with  the  hyperplasia  of  the  abdominal  lymph  glands, 
Peyer's  patches,  and  spleen  which  occurs  in  the  disease,  regarding 
them  as  conservative,  defensive  phenomena,  rather  than  the 
pathological  effects  of  the  bacillus. 

After  a  latent  period  or  negative  phase  of  a  few  days  (the  dura- 
tion being  dependent  on  the  severity  of  the  infection),  the  antibodies 
begin  to  make  their  appearance.  At  first  put  out  only  in  small 
quantities,  they  will  be  absorbed  at  once  by  the  bacilli  present,  and 
will  not  be  demonstrable  in  the  serum  or  plasma  in  a  free  state. 
A  race  now  ensues.  On  the  one  hand,  the  typhoid  bacilli  multiply 
as  rapidly  as  their  environment  will  allow ;  on  the  other,  the 
immune  body  and  other  defensive  antibodies  are  being  gradually 
elaborated  more  and  more  rapidly.  In  a  very  severe  infection 
this  latter  process  may  perhaps  make  default  altogether,  and  the 
patient  succumb  during  the  negative  phase.  This  has  not  been 
demonstrated  in  the  case  of  the  bacteriolytic  substances,  but  is 
well  known  in  the  case  of  the  agglutinins.  In  most  cases,  how- 
ever, the  increase  of  bacteria  and  of  antibodies  takes  place  at  the 
same  time.  After  a  time  bacteriolysis  occurs,  but  in  small 
amount,  since  but  little  immune  body  makes  its  appearance,  and 
that  which  is  formed  at  first  is  probably  utilized  as  opsonin. 
Hence  when  solution  of  the  bacteria  and  liberation  of  the  toxins 
does  occur,  it  does  so  at  first  only  to  a  small  extent,  and  we  may 
regard  it  probable  that  the  minute  amount  of  endotoxin  set  free 
does  not  add  appreciably  to  the  symptoms  of  intoxication  already 
present.  Further,  we  have  now  the  conditions  under  which 
immunity  to  these  toxins  may  be  expected  to  develop :  the  setting 
free  of  small  but  gradually  increasing  doses  of  endotoxin — condi- 
tions, in  fact,  closely  similar  to  those  obtaining  in  Macfadyen's 
experiments.  Probably,  therefore,  by  the  time  that  much  bacterio- 
lysis occurs  the  patient's  tissues  are  more  or  less  immunized.  It 
is,  however,  not  unlikely  that  the  rapid  oscillations  of  temperature 
characteristic  of  the  latter  stages  of  typhoid  fever  may  be  due  in 
part  to  this  liberation  of  endotoxin. 

Some  researches  by  Bail  would  appear  to  indicate  that  there  is 
a  sort  of  automatic  mechanism  for  the  prevention  of  bacteriolysis 
in  the  tissues.  He  found  that  no  bacteriolysis  took  place  when  a 
powerful  serum  was  mixed  with  emulsions  of  cells  from  the  liver, 
spleen,  etc,  as  well  as  with  bacteria.  The  explanation  is  doubtless 
that  the  complement  is  absorbed  by  these  cells,  as  has  been  pointed 


ON    IMMUNITY   TO    BACTERIA  341 

out  by  Hoke,  Muir,  and  others.  Now,  if  this  process  goes  on  in 
the  living  body,  it  can  only  indicate  that  complement  (and  thermo- 
labile  opsonin)  can  never  occur  in  a  free  state  in  the  organs,  and 
hence  that  bacteriolysis  does  not  occur  in  these  regions.  It  must 
be  pointed  out,  however,  that  amboceptor  (or  thermostable  opsonin) 
is  not  absorbed  in  this  way,  so  that  there  would  be  but  little  inter- 
ference with  phagocytosis.  It  is  quite  possible  that  this  absorp- 
tion of  complement  does  take  place  in  order  to  avoid  the  liberation 
of  endotoxin  consequent  on  bacteriolysis. 

The  mobilization  of  the  patient's  defences  is  usually  manifested 
by  an  increase  in  the  number  of  the  leucocytes  in  the  circulating 
blood.  This  subserves  two  functions  :  there  is  a  greater  number 
of  leucocytes  to  act  as  phagocytes  and  there  is  an  increase  in  the 
possible  number  of  cells  which  may  produce  alexin  or  complement. 
Hence  we  find  leucocytosis  present  almost  invariably  in  cases  in 
which  bacteria  gain  access  to  the  blood;  the  exceptions  being, 
firstly,  very  severe  infections  in  which  the  defensive  reaction  fails, 
and,  secondly,  a  group  of  diseases  of  which  typhoid  fever  is  the 
best  example.  The  meaning  of  the  first  exception  is  tolerably 
clear,  that  of  the  second  not  at  all  apparent. 

The  mechanism  by  which  this  increased  output  of  leucocytes  is 
produced  is  chemotaxis.  The  bacteria  in  the  blood-stream  produce 
a  toxin  which,  in  comparatively  mild  infections  or  in  severe 
infections  in  an  animal  possessed  of  strong  natural  immunity, 
attacks  the  leucocytes.  These  cells  will,  therefore,  tend  to  leave 
the  region  in  which  they  are  formed  and  in  which  they  are  lying 
dormant  and  make  their  way  into  the  blood,  the  amount  of  toxin 
being  greater  in  the  latter  than  the  former  situation.  At  the  same 
time,  as  Muir  has  so  conclusively  shown,  there  is  an  increase  in 
the  functional  activity  of  the  bone-marrow,  which  manifests  itself 
in  an  increase  of  the  leucocyte  (and  especially  polynuclear 
leucocyte)  producing  cells.  A  double  process  goes  on,  leucocytes 
being  formed  more  rapidly  and  attracted  from  the  bone-morrow 
as  soon  as  they  are  produced.  That  this  is  due  to  chemotaxis  is 
shown  by  the  fact  that  it  follows  the  injection  of  bacterial  toxins 
and  other  products  into  the  blood-stream,  if  not  too  virulent  or  in 
too  large  amount. 

When  either  of  these  conditions  occurs  we  see  the  opposite 
result — a  leucopaenia,  or  at  least  an  absence  of  leucocytosis,  and 
this  is  always  an  extremely  bad  omen  when  it  occurs  in  those 
infectious  processes  where  leucocytosis  ordinarily  takes  place. 


342  VIRULENCE    OF    BACTERIA 

Thus  in  pneumonia  the  number  of  leucocytes  per  cubic  millimetre 
has  a  most  important  prognostic  value,  and  in  almost  all  cases  it 
will  be  found  that  a  patient  with  a  high  leucocytosis  will  recover, 
even  if  the  attack  is  a  severe  one  and  the  clinical  signs  unfavour- 
able. This  leucopaenia  may  be  attributed  either  to  negative 
chemotaxis,  or  to  the  production  of  paralysis  or  death  of  the 
leucocytes,  or  to  inhibition  of  the  functions  of  the  bone-marrow, 
or  to  a  combination  of  these  causes.  In  any  case,  it  argues  a  high 
grade  of  virulence  on  the  part  of  the  bacteria,  which,  by  lessening 
the  action  of  the  main  natural  protective  forces,  increases  the 
chance  of  a  lethal  issue  to  the  disease.  Let  us,  therefore,  discuss 
some  of  the  main  facts  (many  of  which  have  been  referred  to 
before)  concerning  the  nature  of  the  mechanism  of  an  increase  in 
virulence. 

For  an  organism  to  be  virulent  it  must  produce  a  toxin  which 
has  a  profound  action  on  the  cells  (and,  it  may  be  added,  on  the 
important  cells)  of  the  animal  in  question.  This,  of  course,  is 
obvious,  and  without  it  the  organism  could  only  produce  pathogenic 
effects  by  such  means  as  deprivation  of  oxygen  or  food-stuffs  from 
the  host  or  the  production  of  bacterial  emboli — results  which  are 
of  no  practical  importance.  Without  an  active  toxin  the  bacteria 
would  either  lie  latent,  as  occurs  in  typhoid  fever,  gonorrhoea  and 
tubercle,  or  circulate  in  the  blood  as  a  harmless  parasite,  as  is  the 
case  with  many  protozoa  in  the  lower  animals. 

As  regards  the  special  actions  of  toxins  which  render  the  bacteria 
especially  virulent,  we  may  distinguish  two — (i)  a  leucocytic  or 
leucolytic  action,  and  (2)  negative  chemotaxis.  The  former  is  a 
very  common  function  of  highly  virulent  bacteria;  its  action  is 
best  seen  in  the  production  of  pus  and  necrosis  in  local  lesions, 
but  its  presence  may  be  inferred  from  the  degenerative  changes 
frequently  present  in  leucocytes  in  general  infections.  And  these 
leucocytic  substances  need  not  be  true  toxins,  for  there  is 
reason  to  believe  that  the  non-specific  enzymes  and  other 
substances  which  the  bacteria  produce  have  this  action,  and 
that  the  death  and  destruction  of  leucocytes  which  is  so  marked 
a  feature  of  abscess  production  may  be  due  in  part  to  their 
action. 

We  have  already  referred  to  the  question  of  negative  chemotaxis. 
It  probably  actually  occurs,  but  the  proof  is  hardly  convincing, 
and  the  subject  needs  investigation  by  modern  methods  in  vitro. 
There  is  no  doubt,  however,  that  in  virulent  infections  the  regions 


ON    IMMUNITY   TO    BACTERIA  343 

containing  the  bacteria  are  often  singularly  free  from  leucocytes, 
be  the  explanation  what  it  may. 

The  first  necessity  for  virulence,  therefore,  is  the  possession  of 
an  active  toxin,  which,  either  by  lowering  the  general  vitality, 
or  by  repelling,  paralyzing,  injuring,  or  killing  the  leucocytes, 
diminishes  the  amount  of  phagocytosis  which  occurs. 

A  second  defence  against  phagocytosis  is  the  production  of  a 
defensive  envelope.  This  is  well  seen  in  the  case  of  virulent 
anthrax  bacilli,  which  form  thick  gelatinous  envelopes  in  the 
animal  body.  Such  capsulated  forms  are  only  taken  up  with 
difficulty  by  the  leucocytes,  and  the  greater  the  power  of  capsule 
formation  the  greater  the  virulence  of  the  culture.  Similar 
phenomena  have  been  seen  in  the  case  of  the  streptococci, 
tubercle  bacilli,  and  other  organisms.  The  capsule  of  the 
pneumococcus,  which  is  only  developed  in  the  animal  body  or  in 
culture  fluids  approximating  thereto,  is  probably  a  case  in  point. 

Capsule  formation  is  also  probably  a  defence  against  bacterio- 
lysins.  Thus,  according  to  Eisenberg,  virulent  typhoid  or  coli 
bacilli  stain  blue  by  Giemsa's  method,  and  show  a  pink  edge. 
Organisms  which  have  undergone  this  modification  are  suscep- 
tible to  phagocytosis,  but  show  great  resistance  to  bacteriolysis. 
And  it  is  probable  that  the  capsule  of  the  anthrax  bacillus  is 
intended  largely  as  a  defence  against  bacteriolytic  substances.  The 
bacilli  can  be  trained  to  produce  it  by  cultivation  in  the  serum  of 
the  rat,  which  dissolves  the  unaltered  bacilli  in  large  numbers. 

A  third  and  more  subtle  method  in  which  a  bacterium  can 
increase  its  virulence  is  by  loss  of  the  receptors  (which  it  possesses 
in  the  avirulent  state),  which  have  the  power  of  anchoring  the 
defensive  substances  of  the  blood.  Thus  the  virulent  pneumo- 
cocci  which  (as  shown  by  Rosenau)  are  not  ingested  by  leucocytes 
in  presence  of  serum  escape,  as  a  result  of  the  fact  that  they  do 
not  absorb  opsonin.  Typhoid  bacilli  which  have  been  cultivated 
in  immune  serum  lose  their  power  of  combining  with  amboceptor, 
and  also  with  agglutinin,  and  it  may  be  taken  as  a  general  rule 
that  the  more  virulent  a  culture  the  less  its  reaction  to  its  agglu- 
tinin, and  the  less  it  absorbs  that  substance.  Thus  typhoid  bacilli, 
when  isolated  from  the  body,  are  usually  refractory  to  agglutina- 
tion, and  gain  the  property  only  after  several  generations  of  culture 
on  artificial  media. 

Hence,  therefore,  we  may  picture  the  process  which  goes  on 
when  a  dose  of  pathogenic  bacteria  gains  access  to  the  blood  of  a 


344       SEQUENCE    OF    EVENTS    IN    GENERAL    INFECTIONS 

susceptible  animal  somewhat  as  follows  :  The  bacteria  form  a 
toxin  which  attracts  the  leucocytes  from  the  bone-marrow  into 
the  blood,  and  which  also  stimulates  their  production  in  larger 
numbers.  It  also  attracts  the  leucocytes  into  the  neighbourhood 
of  the  bacteria,  and  this  process  is  more  marked  in  regions,  such 
as  the  spleen  and  bone-marrow,  in  which  the  flow  is  slow.  As  a 
result  some  leucocytes  are  stimulated  to  produce  complement,  or, 
on  the  other  theory,  are  killed,  and  their  complement  set  free.  In 
either  case  the  bacteria  are  prepared  for  phagocytosis.  This 
process  may  now  take  place,  the  bacteria  be  destroyed,  and  the 
infection  come  to  an  end.  This  is  a  type  of  a  mild  infection. 

Or  it  may  happen  that  the  toxin  is  so  powerful  that  the  leuco- 
cytes are  repelled,  or,  if  attracted,  immediately  killed.  If  this 
occurs  the  sole  resource  of  the  patient  is  the  bacteriolytic  property 
of  the  blood,  which  will  then  come  into  action — provided,  of  course, 
that  the  bacterium  is  one  to  which  amboceptor  occurs  in  the  normal 
blood — because  alexin  is  set  free  in  the  solution  of  the  leucocytes. 
Experiment,  however,  leads  us  to  believe  that  this  is  a  slower 
and  less  important  process  than  phagocytosis,  and  that  if  the  latter 
is  in  abeyance  the  outlook  for  the  patient  is  bad  in  the  extreme. 
If  both  processes  fail  the  infection  must  be  rapidly  fatal. 

In  an  infection  in  which  the  defensive  and  offensive  mechanisms 
are  nicely  balanced,  and  an  illness  of  some  duration  and  severity 
occurs,  the  processes  will  be  much  more  complex.  The  early 
stages  will  occur  as  in  a  mild  infection,  and  the  bacteria  will  be 
surrounded  by  the  leucocytes,  and  perhaps  even  ingested.  In  this 
case  it  may  happen  that  after  ingestion  solution  will  take  place, 
and  an  endotoxin  be  set  free  which  will  kill  the  phagocyte,  and 
perhaps  also  those  in  the  immediate  neighbourhood,  and  in  this 
way  some  of  the  bacteria  may  be  shielded  from  further  phagocy- 
tosis for  a  time.  This  and  similar  processes  give  the  bacteria 
time  in  which  to  undergo  their  defensive  modifications,  which  they 
do  in  virtue  of  the  "  survival  of  the  fittest  " — i.e.,  those  which  are 
best  adapted  to  their  environment  in  the  host.  Now  it  is  clear 
that  in  any  culture  of  bacteria  the  different  individuals  have 
marked  differences  with  regard  to  their  power  of  absorbing  anti- 
bodies. This  is  obvious  when  we  consider  that  otherwise  the 
addition  of  gradually  increasing  amounts  of  an  agglutinating 
serum  would  cause  no  effect  until  a  certain  concentration  was 
reached,  when  all  the  bacteria  would  clump.  As  it  is,  those 
organisms  with  the  greatest  affinity  take  up  the  agglutinin  in 


ON    IMMUNITY    TO    BACTERIA  345 

largest  quantity,  and  when  the  number  of  molecules  reaches  a 
certain  amount  they  clump,  whilst  those  with  less  affinity  have 
not  yet  absorbed  sufficient.  Probably  exactly  similar  phenomena 
occur  in  the  opsonization  of  bacteria.  Some  organisms  are  avid 
for  opsonin ;  others  take  it  up  with  difficulty,  and  a  large  concen- 
tration of  the  substance  is  necessary  before  they  are  prepared  for 
phagocytosis.  This  probably  explains  the  fact  that  the  number  of 
bacteria  taken  up  by  the  leucocytes  does  not  increase  pari  passu 
with  the  amount  of  opsonin  present. 

The  bacteria  which  remain  immune  to  the  defensive  mechanisms 
will  be  enabled  to  live,  and  in  their  descendants  the  conditions  for. 
natural  selection  will  occur — variations  and  an  adverse  environ- 
ment. The  weaker  forms — i.e.,  those  which,  by  the  absence  of  a 
defensive  layer  or  .the  presence  of  numerous  receptors  on  which 
the  opsonins  or  bacteriolysins  of  the  host  can  seize,  or  those 
which  do  not  elaborate  a  powerful  toxin — will  be  destroyed,  and 
the  more  virulent  forms  will  survive,  so  that  a  gradual  selection 
of  the  latter  will  ensue,  and  the  invaders  rapidly  increase  in 
virulence.  And  it  must  not  be  forgotten  that  bacteria  multiply 
with  great  rapidity,  division  sometimes  occurring  in  a  quarter  of 
an  hour,  so  that  nearly  a  hundred  generations  are  passed  through 
in  a  day,  and  abundant  opportunity  for  evolutionary  selection 
occurs.  From  what  we  know  of  the  virulence  of  typhoid  bacilli 
and  of  pneumococci  when  causing  disease  and  when  living 
parasitically  without  the  body,  there  is  reason  to  believe  that  this 
increase  of  virulence  always  takes  place  in  infections. 

Here,  then,  the  standing  forces  of  the  body  are  insufficient  to 
deal  with  the  invader,  and  the  latter  increases  in  virulence  and 
numbers  during  the  early  stages  of  the  struggle.  This  process 
will  continue  until  the  latent  reserve  forces  are  mobilized  and 
fresh  defensive  substances  brought  into  action.  These  are 

(1)  antitoxins,  whether  to  exotoxins  or  endotoxins,  or  the  toxins 
may  be  dealt  with  in  one  or  other  of  the  methods  discussed  under 
Antitoxic  Immunity ;  but  in  any  case  it  is  probable  that  the  develop- 
ment of  some  degree  of  toxic  immunity,  especially,  perhaps,  of 
the   leucocytes,  must   precede   the   destruction   of   the   bacteria. 

(2)  Increased  amounts  of  alexin-opsonin  having  a  specific  action 
on  the  organism  in  question.     We  have  already  noted  some  of 
the  gaps  in  our  knowledge  of  this  subject,  and  shall,  perhaps, 
again   have   occasion   to   refer   to   the  difficulties   in   explaining 
its   action,  but   there   can   be  little  doubt  of  its  importance   in 


346  LOCAL    LESIONS 

generalized  infections.  We  notice,  for  example,  that  in  pneumonia 
it  remains  at  a  low  level  during  the  attack,  and  increases  rather 
suddenly  at  the  time  of  the  crisis  ;  the  pneumococci  disappear 
from  the  blood  at  this  period,  though  they  remain  living  in  the 
lung  until  much  later.  In  cases  which  recover  by  lysis,  on  the 
other  hand,  there  is  a  gradual  increase  in  the  opsonic  index,  and  in 
acutely  fatal  pneumococcal  septicaemia  the  index  falls  gradually 
and  continuously.  When  dealing  with  generalized  infections  the 
opsonic  index  (as  far  as  our  researches  have  gone  at  present)  may 
be  taken  as  a  very  rough  guide  to  the  degree  of  immunity  and 
chance  of  recovery.  This  does  not  apply  in  local  infections,  but 
here  we  are  probably  justified  in  believing  that  the  higher  the 
index,  the  less  the  chance  of  generalization.  It  is  worthy  of 
notice  that  the  disease — cerebro-spinal  meningitis — in  which  the 
highest  indices  (sometimes  as  high  as  ten)  are  found  is  one  in 
which  bacteriaemia  very  rarely  occurs,  though  by  analogy  with 
other  similar  diseases  we  should  rather  expect  it  to  do  so.  As 
regards  the  source  of  this  increased  amount  of  alexin-opsonin, 
there  is  nothing  to  be  added  to  what  has  been  already  stated. 
(3)  In  some  cases  recovery  may  be  brought  about  by  the  produc- 
tion of  thermostable  opsonins,  whether  amboceptor  (as  appears 
most  probable)  or  agglutinin.  This  is  rarely,  if  ever,  the  case  in 
the  pneumococcic  diseases,  but  probably  occurs  constantly  in  the 
maladies,  like  typhoid  fever  and  cholera,  in  which  agglutinins  and 
bacteriolysins  are  formed  in  large  amount.  We  may,  indeed, 
classify  diseases  roughly  into  two  main  groups  in  this  respect : 
(i)  those  in  which  the  acquired  immunity  is  due  (inter  alia)  to  the 
presence  of  thermolabile  opsonins  :  in  these  we  may  expect  the 
degree  of  the  immunity  to  be  but  slight  and  its  duration  short; 
and  (2)  those  in  which  it  is  due  to  true  antibodies :  here  we  may 
expect  the  opposite  conditions  to  hold.  Pneumonia  and  typhoid 
fever  may  be  taken  as  examples.  We  do  not  know  accurately 
the  duration  of  the  immunity  to  the  latter  disease,  but  the  anti- 
bodies on  which  we  believe  it  to  depend  may  be  traced  for  long 
periods,  whereas  the  blood  returns  to  its  normal  state  very  shortly 
after  recovery  from  a  pneumococcic  infection.  And  experience 
with  typhoid  vaccine  leads  us  to  suppose  that  the  immunity  to  that 
disease  lasts  a  year  or  more.  The  occurrence  of  relapses  may 
seem  to  argue  that  the  protection  is  in  reality  very  evanescent, 
but  other  interpretations  are  possible. 

The    processes    which    occur    in    local    lesions    are    similar    in 


ON    IMMUNITY    TO    BACTERIA  347 

nature,  but  modified  by  their  occurrence  in  the  tissues  and  not  in 
a  fluid  medium,  and  they  tend  to  deviate  more  from  test-tube 
conditions  than  do  the  blood  infections. 

At  the  commencement  of  the  infection  the  conditions  are  quite 
comparable  to  those  of  a  haemic  infection.  Leucocytes  are  soon 
attracted  into  the  area,  and,  if  the  bacteria  are  not  too  virulent, 
may  remove  them  entirely,  the  infection  being  thus  cut  short  at 
its  very  commencement.  Probably  myriads  of  slight  wounds  are 
thus  dealt  with.  But  when  the  bacteria  resist  phagocytosis,  and 
form  virulent  toxins,  a  new  set  of  factors  are  brought  into 
existence.  These  have  been  glanced  at  previously,  and  will  now 
require  further  discussion. 

The  dilatation  of  the  vessels  of  the  infected  region  and  the 
consequent  acceleration  of  the  blood-flow  is,  as  has  been  pointed 
out,  altogether  a  conservative  influence,  having  for  its  object  the 
removal  of  toxins  and  their  dilution  in  the  general  mass  of  the 
blood.  But  in  all  cases  except  the  most  trivial  the  local  reaction 
extends  further,  and  the  more  extensive  changes  tend,  partly  at 
least,  to  favour  the  spread  of  the  infection  by  shielding  the 
bacteria  from  the  full  action  of  the  blood.  In  other  words,  the 
middle  stages  in  the  evolution  of  an  inflammatory  lesion  indicates 
a  victory  for  the  bacterium  in  so  far  that  it  has  succeeded  in 
altering  the  tissues  to  such  an  extent  as  to  shield  itself  from  the 
action  of  the  protective  forces.  At  a  still  later  stage  (in  lesions 
going  on  to  recovery)  the  tissues  and  juices  gain  the  victory,  and 
there  is  an  alteration  in  the  nature  of  an  increase  in  the  local,  and 
usually  of  the  general,  immunity. 

Let  us  trace  what  we  know  of  this  process  in  the  case  of  a 
small  staphylococcic  lesion.  The  early  stages  consist  of  the  usual 
inflammatory  reaction.  The  increased  access  of  blood,  increased 
number  of  leucocytes,  and  probably  increased  amount  of  opsonin, 
present  in  the  tissues  may  suffice  to  cut  short  the  process,  the 
whole  of  the  bacteria  being  removed  by  phagocytosis.  In  this 
case  the  only  clinical  signs  will  be  those  of  acute  inflammation, 
resulting  in  rapid  recovery. 

If,  however,  the  bacteria  are  present  in  too  great  numbers,  or 
are  too  violent,  or  if  the  immunity  (local  or  general)  is  deficient, 
the  toxins  formed  will  kill  the  tissues  in  the  neighbourhood,  and 
the  bacteria  will  then  lie  in  a  smaller  or  larger  mass  of  necrotic 
tissue,  which  is  surrounded  by  an  inflammatory  zone.  The  tide 
has  now  turned  very  definitely  in  favour  of  the  bacteria,  for  four 


348  SEQUENCE    OF   EVENTS    IN    LOCAL    LESIONS 

reasons :  (i)  There  is  a  mechanical  obstacle  to  the  access  of 
leucocytes  to  the  bacteria,  which  make  their  way  through  the 
slough  with  some  difficulty.  In  ordinary  healthy  tissues,  permeated 
by  capillaries,  the  leucocytes  have  never  very  far  to  go  to  reach  a 
given  area;  but  when  death  of  these  tissues  has  occurred,  the 
leucocytes  have  to  crawl  in  from  the  still  patent  capillaries  of  the 
periphery,  a  distance,  maybe,  of  several  millimetres.  (2)  The 
bacteria  in  the  centre  of  the  lesion  have  time  to  elaborate  a 
powerful  toxin,  which  is  not  diluted  by  the  blood  or  lymph,  since 
the  vessels  are  all  thrombosed,  and  can  only  escape  by  diffusion. 
As  a  result,  there  is  a  central  zone  where  the  toxin  is  present  in  so 
high  a  concentration  that  it  may  either  repel  the  leucocytes  by 
negative  chemotaxis  or  kill  them  outright.  The  latter  process 
certainly  takes  place,  as  the  large  number  of  dead  bacteria  present 
in  pus  sufficiently  proves.  (3)  There  is  an  insufficient  supply  of 
opsonin,  and  perhaps  of  other  defensive  substances  also,  in  the 
fluids  at  the  centre  of  the  lesion.  This  is  brought  about  partly  by 
removal  of  these  substances  by  the  bacteria  of  the  region.  In  the 
case  under  consideration  the  staphylococcus  opsonin  is  absorbed 
by  the  cocci  which,  in  the  absence  of  the  leucocytes,  it  is  power- 
less to  injure.  Another  possible  factor  in  the  removal  of  these 
substances  is  the  action  of  proteolytic  enzymes,  the  defensive 
materials  being  digested  and  rendered  inert  before  they  reach  the 
bacteria.  Lastly,  there  is  reason  to  think  that  opsonins  make 
their  way  with  difficulty  through  the  inflamed  tissues.  Thus 
Bulloch  found  that  the  serum  or  liquor  puris  which  was  collected 
from  an  abscess  immediately  after  it  had  been  cleansed  was  almost 
devoid  of  opsonic  power,  and  this  is  the  case  with  the  fluid  portion 
of  pus  in  general.  In  some  cases  I  have  been  able  to  demonstrate 
the  existence  of  an  antiopsonin,  perhaps  consisting  of  cast-off 
receptors  of  the  bacteria.  (4)  There  is  an  increase  in  the  virulence 
of  the  bacteria,  due  to  conditions  already  discussed. 

The  absence  of  opsonin  from  the  fluid  at  the  centre  of  the 
lesion  suggests  several  considerations  of  some  importance.  We 
see,  for  instance,  that  it  is  not  sufficient  in  the  cure  of  a  local 
lesion  for  there  to  be  an  abundant  supply  of  opsonin  in  the  blood  : 
two  other  factors  are  required— a  permeable  lesion  and  a  region 
suited  for  the  activity  of  leucocytes.  This  is  very  well  seen  in  the 
case  of  tubercle,  where  the  opsonic  index  may  be  greatly  raised  (to 
i '8  or  more),  and  the  lesion  show  no  indication  of  recovery.  There 
is,  as  a  rule,  little  or  no  tendency  of  the  disease  to  spread  or 


ON    IMMUNITY    TO    BACTERIA  349 

become  generalized  with  a  high  opsonic  index,  yet  even  this  may 
occur.  We  may  trace  a  certain  degree  of  parallelism  between  the 
ease  of  access  of  opsonin  to  all  parts  of  the  local  lesion  and  the 
likelihood  of  a  cure  following  an  elevation  of  the  opsonic  index. 
For  example,  there  is,  according  to  TunniclifFe,  a  definite  relation 
between  the  rise  of  the  opsonic  index  and  the  disappearance  of  the 
membrane  in  diphtheria,  the  latter  clearing  rapidly  when  the  index 
rises  above  normal.  Here  the  conditions  are  these  :  there  is  a 
membrane,  a  few  millimetres  thick,  composed  of  dead  material,  in 
which  the  specific  bacilli  are  elaborating  their  toxins ;  below  this 
there  is  a  zone  of  acute  inflammation,  hyperaemic  and  infiltrated  with 
phagocytes.  Now  it  is  easier  for  the  toxins  to  diffuse  outward  and 
be  washed  away  by  the  secretions  of  the  mouth  than  to  pass 
inwards,  and  probably  but  a  small  fraction  actually  formed  reaches 
the  blood-stream.  On  the  other  hand,  it  is  comparatively  easy  for 
the  protective  substances  in  the  serum  to  pass  outwards,  the  con- 
ditions being  quite  different  from  those  in  a  closed  abscess,  where 
opsonins,  etc.,  can  only  reach  the  centre  of  the  lesion  by  diffusion, 
and  not  by  actual  transudation.  Here  there  is  a  constant  stream  of 
lymph  from  the  pervious  vessels  to  the  free  surface.  Thus  the 
low  concentration  of  the  toxins  renders  it  easy  for  the  leucocytes 
to  gain  access  to  the  bacilli,  and  the  latter  have  abundant  oppor- 
tunities of  becoming  opsonized  ;  hence  the  conditions  for  successful 
phagocytosis  are  produced. 

A  similar  train  of  phenomena  follows  the  free  drainage  of  an 
acute  abscess.  The  toxins  escape  outwards,  and  are  no  longer 
forced  into  the  tissues,  and  at  the  same  time  there  is  a  flow  of 
protective  lymph  into  the  abscess  cavity,  and  consequent  sensitiza- 
tion  of  the  bacteria,  and  the  removal  of  the  toxins  allows  the 
phagocytes  to  act.  If,  however,  the  wall  of  the  abscess  is  very 
thick  and  impermeable,  it  may  be,  as  in  Bulloch's  experiment, 
that  the  lymph  which  exudes  is  deprived  of  its  opsonin  and  other 
protective  substances  during  filtration,  and  the  conditions  are  then 
less  favourable. 

Now  consider  a  large  staphylococcic  lesion  completely  embedded 
in  the  tissues.  There  is  a  large  core  of  dead  material,  in  which  the 
cocci  constantly  produce  toxins,  and  in  which  they  are  completely 
shielded  both  from  fresh  lymph  or  plasma  and  from  leucocytes. 
Here  we  should  expect  the  lesion  to  be  progressive,  no  matter  how 
high  the  opsonic  index,  and  this  is  usually  the  case.  Hence,  other 
things  being  equal,  it  is  in  the  smaller  lesions  that  we  may  expect 


350  SEQUENCE    OF   EVENTS   IN    LOCAL   LESIONS 

benefit  from  vaccine  injections  which  raise  the  index,  and  more 
especially  in  cases  in  which  there  is  a  free  drain  for  the  toxins — 
ulcers,  open  abscesses,  sinuses,  etc.,  or  in  those  in  which  there  is 
no  dead  material  (slough  or  caseous  matter),  and  the  blood  is 
brought  into  close  contact  with  the  bacteria.  Small  isolated 
tubercles  in  the  iris  often  yield  rapidly  to  tuberculin  injections* 
whereas  large  caseous  glands  are  most  refractory.1  Chronicity  is 
also  an  important  factor  :  a  long-standing  thick-walled  abscess  is 
less  amenable  to  treatment  than  a  small  freshly  formed  boil. 

In  order  to  aid  this  flow  of  plasma  or  lymph  laden  with  pro- 
tective substances  through  the  lesion,  Wright  has  suggested  the 
exhibition  of  substances  such  as  citric  acid  or  leech  extract,  which 
have  the  power  of  diminishing  the  coagulability  of  the  blood.  In 
certain  cases  the  beneficial  effects  of  these  remedies  may  be  most 
striking.  And  the  local  use  of  hot  fomentations,  etc.,  which  dilate 
the  vessels,  has  a  similar  effect ;  in  addition  to  which  they  probably 
aid  phagocytosis  by  raising  the  temperature  of  the  part. 

Next  as  regards  the  other  pre-existing  defensive  substances  of 
the  plasma — the  bacteriolysins.  In  the  case  of  the  staphylococci 
there  is  no  reason  to  think  that  they  are  of  any  importance,  since 
most  authorities  hold  that  there  is  no  proof  that  serum  has  any 
bactericidal  action  on  these  organisms  under  any  circumstances.2 
And  when  the  blood  does  normally  contain  the  amboceptor- 
complement  apparatus,  the  establishment  of  the  lesion  sufficiently 
demonstrates  the  insufficiency  of  this  apparatus  for  defence. 

At  a  later  stage,  supposing  the  pre-existing  defences — opsonin 
and  leucocytes,  etc. — fail  to  bring  about  cure,  a  second  series  of 
factors  come  into  action.  These  are  the  antibodies,  the  chief 
being  antitoxin,  amboceptor,  and  thermostable  opsonin,  the  latter 
being  possibly  either  agglutinin  or  amboceptor,  as  we  have  seen 
already.  These  usually  take  about  a  week  to  be  produced,  and 
may  be  looked  upon  as  the  second  line  of  defence. 

As  regards  their  place  of  production,  this  may  be  remote  or 
local.  The  sites  of  general  production  of  the  antibodies  have 

1  In  saying  this  I  do  not  wish  to  imply  that  the  cure  in  these  cases,  and  after 
the  use  of  vaccines  in  general,  is  brought  about  solely  by  an  increase  in  the 
opsonins  of  the  blood, 

2  I  must  point  out,   however,   that  in  old  staphylococcic  abscesses  it  is 
common  to  find  cocci  which  have  lost  their  power  of  retaining  Gram's  stain, 
an  invariable  proof  (where  applicable)  of  the  early  stages  of  bacteriolysis. 
This  is  probably  due  to  the  action  of  the  peptic  enzyme  formed  by  the 
leucocytes. 


ON    IMMUNITY   TO    BACTERIA  351 

been  discussed  already,  and  we  have  seen  reason  to  believe  that 
the  chief  regions  in  which  it  takes  place  are  the  lymphoid  organs, 
the  lymph  glands,  bone-marrow,  and  spleen  especially.  These 
organs  appear  to  have  as  one  of  their  functions  the  elaboration  of 
defensive  antibodies  as  an  internal  secretion  in  response  to  toxins 
and  other  bacterial  products  (free  receptors,  etc.)  circulating  in  the 
blood.  We  have  also  seen  that  the  phagocytes,  and  especially  the 
polynuclear  leucocytes,  may  act  as  sources  of  these  bodies,  but 
that  the  evidence  is  less  convincing  than  in  the  case  of  the 
lymphoid  organs.1 

The  local  production  of  antibodies  is  probably  more  important, 
since  it  takes  place  at  the  spot  at  which  these  substances  are 
required.  As  an  example  we  may  take  Romer's  demonstration  of 
antitoxin  in  the  conjunctiva  as  a  result  of  installations  of  abrin, 
when  no  antitoxin  had  yet  appeared  in  the  general  circulation. 
The  source  of  these  antibodies,  as  first  suggested  by  Whitfield,  is 
probably  the  lymphocytes,  which  form  so  important  a  factor  in 
chronic  inflammatory  lesions,  and  which  occur  after  a  few  days  in 
the  periphery  of  acute  lesions.  These  cells  do  not  act  as  phago- 
cytes in  vivo,  though  they  may  do  so  under  experimental  condi- 
tions, and  on  any  other  hypothesis  their  presence  in  inflammatory 
lesions  is  entirely  inexplicable.  We  may  feel  certain,  however, 
that  they  are  attracted  there  for  some  good  purpose,  and  it  is  in 
the  highest  degree  probable  that  this  purpose  is  the  elaboration  of 
protective  antibodies.  It  seems  fairly  certain  that  the  source  of 
the  haemic  antibodies  is  the  lymphocyte  cells  of  the  adenoid 
tissues,  and  the  cells  of  a  chronic  inflammatory  lesion  are  exactly 
similar.  And  in  many  cases  if  we  examine  the  region  in  which 
inflammation  has  taken  place  some  time  previously,  we  shall  find 
that  the  cells  which  were  attracted  thither  have  become  organized 
into  definite  adenoid  tissue. 

It  seems  probable,  too,  that  these  small  round  cells  are  the  basis 
of  the  local  acquired  immunity,  concerning  which  so  little  is 
definitely  known.  After  the  bacteria  have  been  destroyed  the 
polynuclear  leucocytes  remove  the  debris  of  dead  tissues,  etc.,  and 
then  retire,  but  the  lymphocytes  persist  in  the  region  for  long 
periods.  During  this  time  they  are  perhaps  continuously  elaborat- 
ing small  amounts  of  antibodies,  and  are  certainly  on  the  spot  and 
ready  for  action  should  a  fresh  infection  occur.  It  is  probable, 

1  More  recent  researches  tend  to  point  to  the  endothelial  cells  as  the  more 
probable  source  of  origin  of  the  antibodies. 


352  SEQUENCE    OF    EVENTS    IN    LOCAL    LESIONS 

too,  that  they  may  have  become  so  altered  in  virtue  of  having 
once  been  stimulated  to  produce  antibodies  that  they  can  do  so 
more  quickly  and  easily  than  normal  cells.  Thus  Cole  has  shown 
that  an  animal  which  has  once  been  inoculated — e.g.,  with  typhoid 
bacilli — will  produce  on  a  second  injection  a  much  greater  output 
of  antibodies  than  will  a  normal  animal,  and  that  this  increased 
defensive  reaction  persists  for  months,  long  after  the  animal  has 
apparently  become  normal.  This  observation  is  probably  of  the 
highest  importance,  both  as  regards  general  and  local  acquired 
immunity. 

Thus  in  the  case  of  a  local  lesion  we  may  distinguish  two 
stages  :  ( i )  That  in  which  the  natural  resources  of  the  body  alone 
come  into  action,  and  in  which  the  main  mechanism  for  com- 
bating the  bacteria  is  phagocytosis,  the  main  cell  the  polynuclear 
leucocytes,  and  the  main  defensive  substance  the  thermolabile 
opsonin.  In  this  stage  there  is  in  general  a  decline  of  the  local 
immunity  due  to  the  action  of  toxins  on  the  tissues,  and  the  only 
rise  of  the  general  immunity,  if  present  at  all,  is  due  to  an  increase 
of  this  opsonin.  (2)  The  second  stage  is  that  of  the  antibodies. 
The  factors  taking  part  in  the  first  stage  persist,  but  there  is  a 
new  defensive  cell,  the  lymphocyte,  and  new  defensive  substances, 
the  antibodies.  Here  the  immunity,  both  local  and  general,  tends 
to  be  raised.  We  may  also  distinguish  a  third  stage,  in  which  the 
polynuclears  have  retired  and  the  lymphocytes  remain— a  stage  in 
which  the  natural  immunity  of  the  part  is  reinforced  by  the  actual 
or  potential  opsonins  due  to  the  lymphoid  cells,  and  in  which 
these  are  better  equipped  (in  virtue  of  their  previous  training)  to 
produce  a  large  amount  of  antibody  in  response  to  a  slight 
stimulus. 

Lastly,  we  must  study  briefly  the  defensive  reactions  of  the 
peritoneum,  a  region  which  has  been  subjected  to  a  full  investiga- 
tion and  which  resembles  the  tissues  in  some  points  and  the  blood 
in  others.  In  all  probability  the  process  of  absorption  from  the 
other  serous  sacs  is,  in  general,  similar.  We  have  already  glanced 
at  the  subject,  and  in  what  follows  much  use  has  been  made  of 
the  admirable  researches  of  Buxton  and  Torrey.  The  case  of  an 
intraperitoneal  injection  of  a  pathogenic  organism  of  moderate 
virulence  into  an  animal  possessed  of  a  certain  amount  of  natural 
immunity  will  be  considered — e.g.,  of  a  laboratory  culture  of 
B.  typhosus  into  the  peritoneal  cavity  of  the  rabbit. 

Under  these  circumstances,  one  or  other  of  two  trains  of  events 


ON    IMMUNITY   TO    BACTERIA  353 

may  take  place.  In  the  first  the  immunity  appears  to  depend 
mainly  on  bacteriolysis,  in  the  second  on  phagocytosis. 

In  the  former  case  an  enormous  number  of  bacilli  are  killed  in 
the  space  of  a  few  minutes,  very  few  being  recoverable  from  the 
peritoneum  by  washings  with  normal  saline  solution,  and  when 
this  takes  place  it  is  found  that  few  or  no  bacilli  make  their 
way  into  the  blood  or  organs.  In  such  cases  the  typhoid  bacilli 
which  are  recovered  show  marked  signs  of  degeneration,  similar, 
though  less  marked,  to  those  seen  in  cholera  vibrios  in  the  classical 
PfeifFer's  reaction;  the  staining  material  becomes  collected  into 
the  centre  of  the  sheath  of  the  bacillus,  which  subsequently  breaks 
down  into  minute  granules.  This  process  is  usually  entirely 
extracellular,  but  after  a  time  some  of  the  altered  bacilli  may  be 
taken  up  by  the  phagocytes.  It  would  appear  at  first  sight,  there- 
fore, that  in  this  case  the  complement -amboceptor  mechanism 
is  present  in  the  normal  peritoneal  fluid,  ready  to  bring  about 
immediate  bacteriolysis.  This,  however,  is  by  no  means  certain, 
and  it  is  highly  probable  that  the  complement  does  not  exist  in 
this  fluid,  and  that  its  appearance  follows  the  injection,  and  is  due 
in  some  way  to  the  leucopaenia  which  has  been  already  noted. 
The  nature  of  this  leucopaenia  has  been  much  discussed,  and  is  of 
some  importance  in  regard  to  this  question  of  the  nature  of  the 
complement.  Metchnikoff  and  the  French  school  generally 
maintain  that  the  diminution  is  due  to  the  actual  destruction  of 
the  leucocytes— phagolysis — and  that  during  this  destruction  or 
solution  of  these  cells  alexin  or  complement,  or,  as  Metchnikoff 
calls  it,  microcytase,  is  set  free.  As  a  matter  of  fact,  there  is  no 
evidence  that  this  phagolysis  does  occur,  and  more  recent  researches 
appear  to  point  to  another  solution  of  the  leucopaenia.  It  is  true 
that  the  fluid  withdrawn  from  the  peritoneum  by  means  of  a 
pipette  is  markedly  deficient  in  cells,  but  those  that  are  present 
do  not  show  the  marked  degenerative  changes  we  should  expect 
if  they  were  undergoing  rapid  solution.  And  in  any  case,  it  is 
difficult  to  believe  that  an  injection  of  substances  such  as  normal 
saline,  which  we  know  preserves  the  leucocytes  in  a  lively  condition 
for  many  hours,  should  have  such  a  profound  destructive  effect  on 
them  in  the  peritoneum.  Normal  saline,  it  may  be  pointed  out,  is 
capable  of  producing  a  most  marked  leucopaenia  when  injected 
into  the  peritoneal  cavity. 

The  most  probable  explanation  of  the  phenomenon  is  that  first 
suggested  by  Pierallini,  and  corroborated  by  Buxton  and  Torrey. 

23 


354  INFECTIVE    PROCESSES    IN    THE    PERITONEUM 

According  to  them,  there  is  a  deposition  of  masses  of  fibrin  on 
the  omentum,  and  in  and  upon  these  masses  there  are  numerous 
polynuclear  leucocytes. 

The  formation  of  fibrin,  it  need  hardly  be  pointed  out,  is 
universally  regarded  as  being  due  to  the  liberation  of  fibrin 
ferment  by  the  leucocytes.  It  is  not  yet  settled  beyond  controversy 
whether  this  is  to  be  regarded  as  a  process  of  secretion,  a  vital 
phenomenon,  or  a  process  occurring  only  during  the  solution — 
phagolysis — of  the  leucocytes.  The  point  is  of  no  importance  : 
the  remarkable  feature  is  that,  according  to  the  opinions  we  have 
considered  as  most  probably  correct,  complement  is  set  free  at  the 
same  time  as  fibrin  ferment.  The  deposition  of  fibrin  on  the 
surface  of  the  omentum  may  be  taken  as  sufficient  proof  of  the 
liberation  of  this  substance,  so  that  the  rapid  destruction  of  the 
bacteria  which  occurs  in  the  peritoneum  in  some  cases  cannot  be 
regarded  as  constituting  definite  proof  that  it  occurs  preformed  in 
that  situation. 

In  some  cases,  therefore,  bacteria  injected  into  the  peritoneum 
are  killed  rapidly,  almost  instantaneously,  by  a  process  resembling 
bacteriolysis,  and  doubtless  due  to  amboceptor  existing  in  the 
peritoneal  fluid  as  such  at  the  time  of  the  injection,  together  with 
complement  which  is  probably  set  free  from  the  leucocytes  as  a 
result  of  the  presence  of  the  foreign  body,  but  which  may  possibly 
also  be  present  in  the  normal  state.  As  an  example  of  this  process, 
Buxton  and  Torrey  quote  the  case  of  a  rabbit  which  received  an 
intraperitoneal  injection  of  about  4,000,000,000  typhoid  bacilli,  and 
was  then  immediately  killed,  the  peritoneum  washed  out  with 
normal  saline  solution  and  plated  out.  The  fluid  contained  only 
about  1,000  bacilli,  and  there  were  none  in  the  blood  or  internal 
organs.  In  a  case  which  was  allowed  to  survive  for  two  hours  no 
bacilli  were  found  in  any  part  of  the  body.  It  is  most  remarkable 
that,  with  all  this  sudden,  almost  explosive,  solution  of  bacilli  there 
were  no  symptoms  indicative  of  the  liberation  of  endotoxins,  the 
temperature  remaining  constant.  The  culture  was  one  of  very 
moderate  virulence. 

In  other  cases  the  whole  process  differs,  and  the  defence  of  the 
body  appears  to  be  entrusted  to  the  phagocytes  rather  than  to  the 
bacteriolytic  substances,  and  in  these  a  most  interesting  train  of 
phenomena  occurs.  Some  destruction  of  the  bacilli  may  occur  in 
the  peritoneum,  showing  that  the  bacteriolytic  properties  do  not 
fail  entirely,  but  comparatively  few  organisms  are  destroyed  by 


ON    IMMUNITY   TO    BACTERIA  355 

this  process  ;  instead,  large  numbers  of  bacilli  make  their  way 
into  the  circulation  by  a  route  not  fully  known.  These  rapidly 
decrease  in  number,  the  diminution  being  quite  noticeable  within 
half  an  hour,  and  in  six  hours  or  less  the  blood  may  be  entirely 
sterile.  There  is,  further,  a  remarkable  amount  of  deposition  of 
the  bacilli  in  the  organs,  especially  the  liver,  spleen,  lung,  and 
bone-marrow  ;  and  in  these  situations  the  numbers  of  organisms 
decrease  for  about  two  or  three  hours,  and  then  show  a  very 
definite  rise,  which  lasts  for  two  or  three  hours  more,  and  the 
organisms  may  attain  numbers  approximating  to  those  present  in 
these  organs  immediately  after  the  injection.  The  numbers  then 
gradually  decrease  for  the  next  two  days  or  so.  Here  we  may  see 
in  a  very  clear  manner  an  example  of  the  mobilization  of  the 
defensive  forces  discussed  previously.  The  diminution  which 
takes  place  immediately  after  the  access  of  the  bacilli  to  the 
organs  is  doubtless  due  to  the  bacteriolytic  substances,  either 
present  in  the  blood  as  such,  or  immediately  available  after 
phagolysis  or  secretion  of  alexin.  After  a  time  these  substances 
are  exhausted,  and  it  is  only  after  some  hours  that  a  fresh  supply 
is  elaborated,  and  destruction  of  bacilli  continues.  Further,  the 
process  that  now  takes  place  is  mainly  phagocytosis,  whereas  the 
earlier  defensive  process  was  bacteriolysis.  This  sequence  of 
events  is  probably  to  be  interpreted  as  follows  :  As  soon  as  infec- 
tion takes  place  the  small  amount  of  immune  body  naturally 
present  in  the  blood  combines  with  the  bacilli,  and,  since  some 
complement  is  present,  bacteriolysis  occurs.  In  this  way  all  the 
immune  body  is  removed  in  combination  with  the  bacilli,  and  all 
the  complement  is  also  absorbed,  since  the  conditions  for  the 
Bordet-Gengou  phenomenon  are  present,  and  complement  not 
actually  required  for  solution  of  bacilli  will  be  taken  up,  as  well 
as  that  which  is  actually  used  in  the  process.  Hence  all  the 
defensive  substances  are  removed,  and  the  bacilli  can  flourish 
unchecked  for  a  time.  Soon,  however,  more  complement  is 
produced.  This  has  little  or  no  power  of  producing  bacteriolysis  in 
the  absence  of  amboceptor,  but  it  can,  and  does,  act  as  opsonin, 
and  abundant  phagocytosis  occurs.  In  most  diseases  this  stage 
would  be  marked  by  leucocytosis,  but  in  typhoid  fever  this  does 
not  occur.  But  we  shall  shortly  see  that  a  polynuclear  reaction 
does  take  place  in  the  peritoneum,  if  not  in  the  other  regions. 

In  the  series  of  researches  we  are  discussing  the  development 
of  the  last  line  of  research — the  true  antibodies — was  not  studied. 

23-2 


35^  INFECTIVE    PROCESSES    IN    THE    PERITONEUM 

These  only  come  into  action  later.  In  the  case  of  a  mild  infection 
the  whole  process  of  cure  may  occur  without  their  appearance,  and 
they  are  only  of  importance  in  so  far  as  they  are  the  cause  of  the 
subsequent  immunity. 

Let  us  now  turn  to  the  sequence  of  events  in  the  peritoneum  in 
these  animals  in  which  explosive  bacteriolysis  does  not  occur,  or 
only  to  a  small  extent,  in  that  region.  Here  some  phagocytosis  is 
found  to  take  place  in  the  cells  lying  free  in  the  peritoneal  fluid, 
(the  changes  in  which  have  been  noted  previously),  but  the  greater 
part  of  the  process— and  this  is  significant  in  view  of  the  theories 
which  we  have  adopted  with  regard  to  the  liberation  of  comple- 
ment-opsonin  during  the  process  of  coagulation — takes  place  in 
the  masses  of  fibrin  found  on  the  omentum.  At  first  the  bacilli  lie 
free  in  this  deposit,  often  lying  more  or  less  parallel ;  but  in  a 
short  time  vast  numbers  are  taken  up  by  the  macrophages,  and 
very  few  free  organisms  may  be  seen  in  an  hour  or  two  after  the 
injection.  Then  one  of  two  series  of  phenomena  may  occur.  The 
animal  may  recover  ;  and  in  this  case  the  bacilli  which  are  con- 
tained in  the  macrophages  become  pale  and  granular,  and  soon 
disappear  altogether,  and  the  fibrin  becomes  infiltrated  and  eroded 
by  polynuclear  leucocytes.  Or  the  animal  may  die  ;  and  in  this 
case  the  bacilli,  both  those  which  have  been  ingested  and  those 
which  have  not,  commence  to  grow  rapidly,  and  the  organisms, 
after  diminishing  in  numbers  for  a  few  hours,  proliferate  so  quickly 
that  the  animal  dies  in  some  twenty- four  hours.  In  these  cases 
the  bacilli  which  have  been  ingested  are  not  destroyed,  and  retain 
their  normal  appearance  and  staining  reactions  throughout. 
Here  it  is  obvious  that  the  first  line  of  defence,  the  rapid  initial 
phagocytosis,  has  been  unsuccessful,  and  that  the  secondary 
reserve  forces  have  not  had  time  to  come  into  action.  It  is 
specially  pointed  out  by  Buxton  and  Torrey  that  in  these  cases  the 
invasion  of  the  fibrin  by  the  polynuclear  leucocytes  does  not  occur, 
a  fact  strongly  confirmatory  of  the  view  that  it  is  these  cells  that 
give  rise  to  the  opsonin  or  complement,  the  deficiency  in  which 
brings  about  the  lethal  issue. 

Here  we  must  conclude  a  short  and  altogether  inadequate  con- 
sideration of  a  subject  of  the  highest  importance.  Much  more 
might  be  written  on  the  subject,  but  the  very  fact  that  the 
phenomena  do  not  lend  themselves  to  a  brief  discussion  only 
shows  how  very  imperfect  is  our  present  knowledge  of  the  events 
which  actually  take  place  in  the  juices  and  tissues  during  the 


ON    IMMUNITY    TO    BACTERIA  357 

process  of  recovery  from  an  infective  process.  The  mechanisms 
themselves  are  now  fairly  well  known,  the  nature  of  the  substances 
concerned  therein  thoroughly  investigated,  and  their  sources  ascer- 
tained with  some  degree  of  probability;  but  when  we  come  to 
apply  our  knowledge  to  the  actual  events  of  the  living  body,  our 
difficulties  begin  in  earnest.  It  is  in  this  field  of  research  that 
future  advances  are  most  to  be  expected. 

In  conclusion,  let  us  emphasize  the  enormous  importance  which 
we  have  been  led  to  attach  to  the  leucocytes  in  the  struggle  against 
the  infective  diseases.  In  all  probability  the  substance  we  call 
alexin,  thermolabile  opsonin,  or  complement,  and  which,  in  one 
or  other  of  its  actions,  constitutes  the  first  line  of  defence,  is 
formed  by  the  polynuclear  leucocytes.  In  itself  its  action  is  but 
slight,  and  it  requires  to  be  supplemented  either  by  the  leucocyte 
itself,  in  case  the  reaction  is  mainly  phagocytic,  or  by  the  action 
of  amboceptor,  which  we  believe  to  be  derived  from  the  masses  of 
leucocytes  known  as  lymphoid  tissue.  Again,  the  bacterium  may 
give  off  toxin,  especially  if  it  has  escaped  being  taken  up  by  the 
leucocyte,  and  in  this  case  these  cells  exhibit  another  phase  of 
their  protean  activities,  for  that  they  can  actually  absorb  toxin  in 
solution  appears  quite  certain.  If  this  action  fails  the  last  resource 
is  the  neutralization  of  the  toxin  by  antitoxin  ;  and  although  this 
cannot  be  regarded  as  definitely  proved,  there  is  at  least  some 
reason  to  believe  that  this  may  be  formed  in  the  lymphoid  tissues. 
Lastly,  certain  facts  would  seem  to  show  that  it  is  quite  likely 
that  even  the  compound  of  toxin  and  antitoxin  is  by  no  means 
inert  unless  it  has  been  taken  up  by  the  leucocytes,  and  that  the 
action  of  the  latter  substance  is  simply  to  prepare  the  former  so 
that  these  cells  may  attack  it.  In  every  phase  of  the  struggle 
against  the  bacteria  the  leucocyte  appears,  on  certain  or  presump- 
tive evidence,  as  the  particular  cell  to  which  the  defence  of  the 
body  is  entrusted. 


CHAPTER  XIV 

PRACTICAL  APPLICATIONS 
Staphylococcic  Infections. 

COMMON  as  is  disease  due  to  the  staphylococcus  (boils,  carbuncles, 
pustular  acne,  osteomyelitis,  etc.),  our  knowledge  of  the  inner 
mechanism  of  the  pathogenicity  of  this  organism  is  still  somewhat 
scanty.  The  only  toxins  definitely  known  to  exist  are  a  staphylo- 
lysin  and  a  leucocidin.  The  former  is  produced  in  three  or  four 
days  in  faintly  acid  broth,  and  reaches  its  maximum  in  about 
fourteen  days.  It  is  destroyed  by  a  temperature  of  56°  C.,  and  in 
other  respects  resembles  the  true  toxins.  Many  animals,  and 
notably  the  horse,  contain  a  natural  antistaphylolysin,  which 
neutralizes  the  action  of  this  haemolysin,  and  is  apparently  a  true 
antibody.  It  is  present  in  human  serum,  and  may  perhaps 
account  for  the  fact  that  anaemia  is  not  a  striking  feature  of 
Staphylococcic  diseases.  Animals  from  which  it  is  absent,  such 
as  the  rabbit  or  goat,  can  be  made  to  produce  it  by  immuniza- 
tion with  solutions  of  the  lysin.  Leucocidin  is  produced  under 
the  same  conditions  as  the  haemolysin,  and  the  two  are  usually 
produced  side  by  side  ;  but  they  are  distinct  substances,  the  former 
being  the  more  easily  destroyed  by  heat.  Its  action  is  not 
entirely  specific  :  it  kills  the  leucocytes  and  dissolves  them,  but 
has  also  some  action  on  the  ganglionic  and  other  cells.  As  in  the 
case  of  the  hsemolysin,  a  natural  antibody  exists  in  the  serum  of 
man,  the  horse,  etc.,  and  can  be  prepared  from  other  animals. 

These  substances,  especially  perhaps  the  latter,  may  play  some 
role  in  the  production  of  disease,  in  that  they  may  inhibit  phago- 
cytosis by  a  poisonous  action  on  the  leucocytes.  But  there 
is  little  doubt  that  there  is  some  other  toxic  body,  probably  an 
endotoxin,  since  young  cultures  killed  by  heat,  as  used  for  vaccines, 
are  decidedly  toxic,  causing  local  irritation,  and,  if  used  in  large 
doses,  a  rise  of  temperature.  These  vaccines  contain  no  haemo- 
lysin or  leucocidin. 

358 


PRACTICAL  APPLICATIONS  359 

Immunity. — The  resistance  against  staphylococci  appears  to  be 
almost  entirely  dependent  on  phagocytic  action.  Ordinary 
methods  fail  to  demonstrate  any  bactericidal  action,  either  in 
normal  or  immune  serum.  Andrewes  and  Gordon,  however,  by 
the  use  of  special  methods,  have  succeeded  in  showing  that  such 
an  action,  though  very  slight,  occurs  even  in  the  serum  of  normal 
animals,  and  to  a  greater  extent,  though  still  but  slight,  when  the 
animal  has  been  immunized.  Perhaps  the  antibodies  to  the  soluble 
toxins  mentioned  above  may  have  some  protective  action. 

The  staphylococcus  is  very  susceptible  to  phagocytic  action, 
and  there  is  a  general  correlation  between  the  opsonic  index  and 
the  stage  of  evolution  of  the  lesion,  as  shown  in  Fig.  64.  The 
opsonic  index  is  greatly  raised  in  the  serum  of  immunized  animals, 
and  it  would  appear  that  injections  of  dead  staphylococci  have  the 
power  of  increasing  both  the  thermolabile  and  thermostable 
opsonin.  The  duration  of  the  immunity  is  not  definitely  known, 
but  is  certainly  short. 

Diagnosis. — This  is  made  entirely  by  the  demonstration  of  the 
infective  organism — an  easy  task.  In  a  few  cases  the  opsonic 
index  may  afford  some  help,  being  usually  low  during  the  acute 
stage. 

Agglutinating  sera  can  be  prepared,  but  their  action  is  not 
powerful,  and  this  reaction  is  useless  in  diagnosis. 

Treatment. — Antistaphylococcic  sera  have  been  prepared,  but 
the  results  of  its  use  are  not  encouraging,  and  the  only  valid 
method  is  the  use  of  vaccines.  These  should  be  prepared  either 
from  virulent  cultures  derived  from  severe  boils  or  carbuncles 
or  from  the  lesion  it  is  desired  to  treat.  In  practice  it  is  a  good 
plan  to  commence  with  a  stock  vaccine  prepared  from  a  virulent 
culture,  and  to  prepare  an  autochthonous  vaccine  for  use  if  the 
first  does  not  succeed.  In  general  opsonic  control  is  not  necessary, 
and  the  doses  may  vary  between  100  and  1,000  millions,  repeated 
every  ten  or  fourteen  days. 

Streptococcic  Infections. 

The  toxin  of  the  Streptococcus  pyogenes  is  still  not  definitely 
known.  We  have  already  referred  to  the  haemolysin  which  it 
produces.  There  is  some  reason  for  believing  that  this  substance 
has  some  clinical  action  in  acute  infections,  and  that  it  is  produced 
to  a  greater  extent  by  virulent  than  by  non-virulent  cultures. 


360  STREPTOCOCCIC    INFECTIONS 

Apart  from  this,  the  existence  of  a  soluble  toxin  is  doubtful. 
Filtered  broth  cultures  of  the  organism  are  poisonous,  but 
most  observers  have  been^able  to  produce  immunity  therewith, 
and  the  toxic  substances  are  probably  merely  simple  metabolic 
products.  Parascandalo,  it  is  true,  claimed  to  have  been  more 
successful,  but  his  results  have  not  been  corroborated,  and  there 
is  some  reason  to  believe  that  the  nitrate  which  he  used  was  not 
free  from  bacteria,  living  or  dead,  and  that  his  animals  were 
really  vaccinated  with  a  vaccine.  The  toxin  is  probably  an 
endotoxin,  though  of  this  but  little  is  known.  The  bodies  of  the 
bacteria  are  highly  irritating,  and  very  small  doses  of  vaccines 
have  to  be  given  at  the  commencement  of  the  treatment. 

Diagnosis. — This  is  made  by  the  demonstration  of  the  micro- 
organism in  all  cases.  Agglutinating  sera  may  be  prepared, 
but  the  appearance  of  the  property  in  the  serum  in  human 
disease  is  not  sufficiently  marked  or  constant  to  be  of  value. 

Immunity. — Human  blood  appears  to  contain  an  antilysin  which 
neutralizes  the  action  of  streptocolysin,  and  it  is  quite  possible 
that  this  substance  may  play  some  part  in  the  defence  of  the  body 
against  infections.  There  is  also  a  little  evidence  for  the  forma- 
tion of  a  true  antitoxin  against  the  actual  and  efficient  toxin  of  the 
streptococcus,  whatever  this  may  be.  This  is  the  fact  that  in 
some  cases,  though  unfortunately  not  in  all,  the  injection  of  anti- 
streptococcic  serum  is  followed  by  a  marked  immediate  benefit  in 
cases  of  septicaemia,  etc.  The  temperature  may  fall,  the  delirium 
and  headache  pass  off,  and  the  pulse  improve  in  a  striking  manner 
within  an  hour  or  two  of  the  injection.  Now  Wright  has  pointed 
out  that  but  little  is  known  of  the  mode  of  action  of  the  serum, 
and  that  it  may  contain  a  toxic  ingredient  and  really  be  a  vaccine 
in  disguise.  If  this  is  the  case  we  should  not  expect  it  to  produce 
good  effects  so  early  as  actually  happens  in  favourable  cases,  in 
which  its  action  resembles  that  of  diphtheria  antitoxin,  and 
certainly  suggests  that  some  poison  is  neutralized.  Probably, 
therefore,  acquired  immunity  to  streptococci  is  partly  antitoxic  or 
anti-endotoxic. 

Bacterial  immunity  is  probably  mixed,  partly  bactericidal  or 
bacteriolytic  and  partly  phagocytic.  The  serum  of  normal 
animals  has  usually  some  bactericidal  action,  and  this  can  be 
raised  by  immunization  to  a  high  figure  ;  further,  the  serum  of  a 
immunized  animal  is  a  powerful  agent  in  conferring  passive 
immunity,  a  property  not  belonging  (apparently)  to  sera  of  high 


PRACTICAL    APPLICATIONS  361 

opsonic  value.  But  probably  phagocytosis  is  of  the  main  value 
in  the  struggle  against  the  streptococcus  in  the  body.  The 
organisms  are  very  readily  taken  up  by  the  leucocytes  in  presence 
of  serum,  and  are  frequently  seen  inside  the  pus  cells  in  suppura- 
tive  diseases-  In  general  the  opsonic  index  may  be  taken  as  a 
fair  index  of  the  degree  of  immunity,  since  it  is  usually  low  during 
the  course  of  the  infection  and  rises  as  cure  is  brought  about. 
This  has  been  already  shown  in  the  case  of  erysipelas  :  the  index 
returns  to  normal  in  one  to  three  days  after  the  rise,  indicating 
that  the  immunity  in  this  case  is  but  transient  (see  Fig.  63).  This 
is  of  interest  in  view  of  the  frequency  with  which  this  disease  is 
recurrent.  I  have  seen  a  case  in  which  attacks  occurred  every 
three  weeks  for  a  year.  But  the  solid  immunity  conferred  by 
vaccination  and  the  use  of  massive  cultures  is  more  lasting,  due 
probably  to  the  formation  of  immune  bodies  and  thermostable 
opsonins. 

Local  immunity  probably  also  plays  a  part  of  great  importance 
in  streptococcic  infections,  as  shown  by  the  fact  that  the  ery- 
sipelatous  lesion  usually  spreads  at  one  margin  and  heals  at 
another  simultaneously.  Yet  even  here  the  spread  may  be  due  to 
failure  of  access  of  the  blood  to  the  bacteria.  This  is  suggested 
by  the  frequent  success  of  the  local  use  of  iodine  and  other 
rubefacients  in  arresting  its  spread.  A  similar  effect  may  some- 
times be  seen  in  chronic  cases  by  the  use  of  citrates  or  one  or 
other  of  the  means  suggested  by  Wright  for  lowering  the  coagu- 
lability of  the  blood.  A  case  in  which  the  disease  had  been 
present  for  five  weeks  recovered  in  two  days  when  treated  in 
this  way. 

Treatment. — Protective  treatment  is  not  employed.  It  is,  how- 
ever, interesting  to.  note  a  fact  pointed  out  by  Sir  James  Paget 
many  years  ago.  Pathologists  are  as  a  rule  more  or  less  immune 
to  septic  infections,  and  these  comparatively  rarely  occur  in  those 
who  are  in  the  daily  habit  of  performing  post-mortem  examina- 
tions. The  attack  which  proved  so  nearly  fatal  to  him  occurred 
after  a  long  absence  from  the  post-mortem  room.  Probably  most 
pathologists  are  vaccinated  against  the  disease. 

The  curative  treatment  (apart,  of  course,  from  that  of  the 
local  lesion,  if  any,  and  the  use  of  general  toxic  and  sustaining 
measures)  resolves  itself  into  the  question  whether  a  serum  or 
vaccine  should  be  used.  This  question  cannot  be  definitely 
settled  at  the  present  time,  and  the  recommendations  which  are 


362  STREPTOCOCCIC    INFECTIONS 

given  here  may  require  great  alteration  in  the  future.  In  several 
and  rapidly  progressing  septic  cases,  such  as  those  due  to  post- 
mortem wounds,  etc.,  the  use  of  serum  is  indicated,  and  offers  the 
best  chance  of  success.  In  view  of  the  very  great  benefit  which 
is  frequently  derived  from  this  measure,  it  appears  highly  im- 
proper to  wait  until  a  vaccine  is  prepared.  This  should  be  taken 
in  hand  at  once,  so  that  a  homologous  vaccine  may  be  ready  if 
subsequent  events  suggest  its  use,  but  in  the  meantime  serum 
should  be  employed.  The  use  of  minute  doses  of  a  stock  vaccine 
(5  to  10  millions  per  dose)  may  be  considered,  and  there 
appears  to  be  no  reason  why  they  should  not  be  given  along  with 
the  serum.  If  practicable,  opsonic  estimations  should  be  taken, 
but  the  streptococcus  is  not  as  a  rule  a  very  satisfactory  organism 
to  work  with.  In  the  absence  of  such  observations,  doses  of  the 
size  mentioned  above  may  be  given  every  three  or  four  days. 

In  chronic  septicaemia  or  ulcerative  endocarditis  of  streptococcic 
origin  the  use  of  vaccines  offers  more  prospect  of  success,  though 
even  here  cures  have  been  brought  about  by  means  of  the  serum, 
which  should  be  used  whilst  cultures  are  being  taken  from  the 
blood  and  a  vaccine  prepared.  At  present  it  seems  desirable  to 
use  careful  opsonic  control  in  cases  of  this  class.  An  excellent 
illustrative  case  treated  by  Douglas  should  be  referred  to  for 
details.  Here  the  doses  were  5  tc  12  millions,  and  the 
injections  were  given  each  time  the  index  fell.  The  case  was 
certainly  one  of  septicaemia,  but  the  evidence  for  the  existence  of 
ulcerative  endocarditis  is  not  conclusive. 

In  chronic  local  inflammatory  disease  of  streptococcic  origin 
vaccine  treatment  is  probably  the  best,  but  here  ordinary  methods 
(especially  Bier's)  may  be  of  more  advantage.  In  the  absence  of 
opsonic  control,  the  doses  may  begin  at  10  millions  and  rise  to 
100  millions,  and  be  given  once  a  week. 

Erysipelas  does  not  usually  require  specific  treatment,  and  in 
severe  cases  serum  often  answers  well.  The  use  of  vaccines  has 
not  been  tried  on  a  sufficiently  large  scale  to  allow  us  to  judge  of 
its  value.  A  case  under  my  care  of  recurrent  erysipelas,  in  which 
attacks  had  occurred  about  every  three  weeks  with  some  degree 
of  regularity  for  a  year,  was  apparently  cured  by  a  few  doses  of 
vaccine,  commencing  at  25  millions  and  rising  to  50  millions,  and 
administered  once  every  ten  days.  In  some  chronic  cases  of 
erysipelas  it  is  probable  that  there  is  a  considerable  amount  of 
haemic  immunity,  but  the  protective  substances  are  unable  to 


PRACTICAL   APPLICATIONS  363 

reach  the  bacteria.     In  these  the  use  of  citrates  or  citric  acid  (as 
recommended  by  Wright)  is  occasionally  of  great  value. 

The  mode  of  action  of  antistreptococcic  serum  is  still  quite 
uncertain,  and  its  use  is  purely  empirical.  Probably  in  most 
cases  it  acts  as  a  bacteriolytic  substance.  Wright  has  suggested 
that  it  may  contain  free  toxin,  and  so  act  as  a  vaccine ;  but  the 
fact  that  it  will  passively  immunize  animals  treated  with  it  is 
adverse  to  this  supposition.  It  is  prepared  by  gradually  immuniz- 
ing horses  and  other  animals  until  they  can  withstand  large  doses 
of  the  living  organisms.  The  differences  between  the  various 
brands  mainly  arise  from  the  nature  of  the  culture  used  for  the 
purpose.  The  earlier  sera  to  be  prepared  were  procured  by  the 
injection  of  a  single  strain  of  streptococcus  isolated  from  a  case 
of  erysipelas,  puerperal  fever,  etc.  It  was  found,  however,  that 
this  serum  was  not  successful  in  every  case,  and  attempts  were 
made  to  improve  it.  Starting  from  the  supposition  that  the 
reason  for  the  non-success  was  the  fact  that  the  streptococcus 
used  was  not  exactly  the  same  variety  as  that  causing  the  infec- 
tion in  the  patient,  polyvalent  sera  were  prepared  by  treating  horses 
with  cultures  from  many  various  sources,  and  it  is  sera  of  this 
nature  that  are  now  in  general  use.  Another  explanation  was 
that  the  cultures  used  were  not  of  sufficient  virulence,  and  as 
antibodies  to  an  attenuated  organism  may  not  act  against  the 
same  culture  when  the  virulence  is  exalted,  strains  of  great 
potency  were  obtained  by  means  of  passage  through  rabbits.  Yet 
a  third  method  has  been  adopted,  based  on  the  fact  that  an 
organism  which  is  highly  virulent  for  one  animal  may  be  harmless 
to  another  species.  Here  passage  is  avoided,  and  the  cultures 
used  in  the  immunization  of  the  horse  are  taken  direct  from 
human  disease,  and  are  used  soon  after  isolation— «'.£.,  before 
virulence  has  been  lost  by  prolonged  cultivation  in  vitro.  There 
is  no  very  clear  evidence  that  any  special  advantage  attaches  to 
any  of  the  sera  thus  prepared ;  in  any  given  case  it  is  a  matter  of 
chance  which  sample  will  prove  successful.  The  criterion  as  to 
the  suitability  of  a  given  sample  of  serum  is  purely  an  empirical 
one.  The  patient's  pulse,  temperature,  nervous  condition,  etc., 
should  be  watched  with  the  closest  attention,  and  any  definite 
improvement  occurring  within  the  first  twenty-four  (and  usually 
within  the  first  four)  hours  of  the  first  doses  should  be  an  indica- 
tion to  continue  with  the  use  of  the  same  brand  of  serum.  In 
some  cases  each  injection  causes  a  slight  but  definite  rise  of 


364  PNEUMOCOCCIC    INFECTIONS 

temperature,  and  the  symptoms  undergo  a  brief  exacerbation, 
followed  by  an  improvement  in  the  general  condition.  The 
meaning  of  this  phenomenon  is  somewhat  doubtful,  Wright  hold- 
ing that  the  rise  is  due  to  the  presence  of  toxin  in  the  serum.  It 
seems,  however,  more  probable  that  it  is  due  to  a  solution  of  some 
of  the  streptococci  and  consequent  liberation  of  endotoxin.  It  is 
not  a  contra-indication'bto  the  use  of  the  serum,  though  it  may 
suggest  care  in  its  use.  If  no  obvious  result  follows  the  use  of 
the  serum,  the  brand  should  be  changed,  another  specimen  being 
administered  forthwith. 

Antistreptococcic  serum  is,  as  a  rule,  not  standardized,  and  the 
initial  dose  should  not  be  less  than  10  c.c.,  whilst  20  to  25  c.c.  is 
not  too  much.  Subsequently  10  c.c.  may  be  given  each  day  as 
long  as  it  appears  to  be  of  benefit.  In  a  generalized  infection  it 
seems  reasonable  to  give  the  injections  intravenously,  but  there  is 
no  proof  that  the  method  is  of  more  value  than  the  ordinary 
process  of  injecting  it  into  the  cellular  tissue  of  the  flank. 

Good  results  have  been  obtained  in  severe  cases  of  scarlet  fever 
by  the  use  of  serum  obtained  by  the  use  of  streptococci  obtained 
from  scarlatinal  throats  (S.  conglomerate} .  It  appears  tolerably 
clear  in  this  disease  that  a  homologous  serum  is  of  advantage,  the 
results  being  better  than  those  got  by  the  use  of  ordinary  anti- 
streptococcic  serum. 

Pneumococcic  Infections. 

The  marked  remote  toxic  symptoms  frequently  met  with  in 
pneumonia  would  suggest  that  the  pneumococcus  forms  an 
exotoxin,  and  there  is  a  certain  amount  of  experimental  support 
for  this  supposition.  The  Klemperers  made  use  of  nitrates  in 
their  experiments  in  the  production  of  an  antitoxic  serum,  and 
found  them  fairly  toxic,  and  possessed  of  undoubted  immunizing 
powers ;  and  similar  results  have  been  obtained  by  Washbourn, 
Isaeff,  and  others.  But  the  potency  of  these  filtrates  is  slight 
compared  with  that  of  a  true  exotoxin,  and  the  results  obtained 
are  quite  consistent  with  the  supposition  that  they  are  due  to 
slight  autolysis  and  liberation  of  an  endotoxin.  The  fact  that 
lesions  occur  remote  from  the  lung  cannot  be  taken  to  suggest 
that  an  exotoxin  is  produced,  since  we  know  that  in  pneumonia 
considerable  numbers  of  cocci  make  their  way  into,  and  are 
destroyed  in,  the  blood-stream.  Vaccines  of  the  dead  cocci  are 
but  slightly  toxic. 


PRACTICAL  APPLICATIONS  365 

According  to  Casagrandi,  non-pathogenic  varieties  of  the 
pneumococcus  form  a  haemolysin,  whilst  virulent  ones  do  not. 
The  same  observer  states  that  certain  cultures  produce  a  leuco- 
cidin. 

The  nature  of  the  immunity  to  the  pneumococcus,  and  in 
especial  the  causation  of  the  crisis  (when  present),  has  been  much 
discussed,  and  now  seems  to  be  fairly  well  elucidated.  It  is 
doubtful  whether  any.  bactericidal  substances  are  formed  in  man 
after  an  attack  of  pneumonia ;  they  may  be  produced  in  rabbits  as 
a  result  of  artificial  immunization,  but  are  not  invariably  present, 
and  it  is  useless  to  attempt  to  explain  the  characteristic  crisis  by 
their  agency.  Anti-endotoxin  may  perhaps  be  formed  artificially, 
but  its  presence  in  human  disease  is  very  doubtful.  It  is,  how- 
ever, now  tolerably  clear  that  recovery  in  pneumococcic  infections 
is  brought  about  mainly  by  the  action  of  opsonins,  and  more 
especially  thermolabile  opsonins.  This  was  well  shown  by 
Mac  Donald,  whose  investigations  have  been  referred  to  already. 
The  opsonic  index  is  low  during  the  course  of  the  attack,  and  rises 
suddenly  to  normal  at  or  near  the  crisis.  Eyre  distinguishes  three 
types  of  opsonic  index  in  different  forms  of  pneumococcic  infec- 
tions. The  first  is  the  pneumonic  type,  his  results  precisely 
corroborating  Mac  Donald ;  the  second  is  the  form  in  which 
recovery  takes  place  by  lysis,  in  which  the  rise  is  gradual ;  and 
the  third,  in  which  the  disease  progresses  to  a  fatal  termination,  is 
characterized  by  a  steady  fall  in  the  index.  Eyre  holds  that  the 
resistance  of  the  individual  may  be  measured  by  his  opsonic 
response,  as  represented  by  these  three  curves. 

The  opsonin  present  in  natural  disease  is,  as  mentioned  above, 
mostly  thermootablc ;  but  in  the  immunized  sera  of  hypervaccinated 
animals  thermostable  opsonin,  the  bacteriotropin  of  Neufeld  and 
Rimpau,  is  present.  In  all  probability  it  is  to  these  substances 
that  antipneumococcic  serum  owes  its  effects.  These  are  not 
great — according  to  Eyre,  i  c.c.  of  the  most  powerful  serum  yet 
obtained  will  only  protect  against  some  300  lethal  doses — as  we 
should  expect  to  be  the  case  if  the  immunity  conveyed  were  due 
to  a  rise  in  the  opsonic  value  of  the  blood. 

Agglutinins  are  usually  found  in  cases  of  pneumonia,  but  they 
are  not  present  in  large  amount  (the  serum  rarely  clumps  at  a 
greater  dilution  than  i  :  60),  though  a  much  more  powerful  action 
may  be  obtained  by  artificial  immune  serum.  The  reaction  is  of 
little  value  in  diagnosis,  since  it  may  be  absent  even  in  pneumonia, 


366  PNEUMOCOCCIC   INFECTIONS 

and  is  often  absent  in  the  more  localized  infections.  The  disease 
is  recognized  by  the  demonstration  of  the  diplococcus. 

In  acute  pneumococcic  infections  (pneumonia,  septicaemia, 
peritonitis,  etc.)  attended  with  severe  constitutional  disturbance 
the  use  of  serum  may  be  tried.  Several  have  been  prepared,  the 
methods  used  differing  somewhat  in  the  different  cases  ;  but,  with 
the  exception  of  that  prepared  by  the  Klemperers,  the  animals 
used  are  immunized  by  the  use  of  dead,  and  subsequently  of  living, 
cultures.  The  best  known  are  Pane's  and  Romer's.  The  latter  is 
markedly  polyvalent,  an  important  point,  since  various  strains  of 
pneumococci  probably  differ  largely  inter  se.  This  was  very  well 
shown  by  Washbourn  and  Eyre,  who  found  Pane's  serum 
protective  against  four  cultures  from  different  sources,  but  power- 
less against  a  fifth. 

The  results  of  the  use  of  antipneumococcic  serum  have  not 
been  such  as  to  lead  to  its  general  use,  although  several  observers 
have  reported  very  good  effects.  According  to  Tauber,  the  tem- 
perature falls  to  normal  after  one  to  three  injections  of  20  c.c., 
and  there  is  marked  mental  and  general  improvement.  Stress 
is  laid  on  this  by  other  physicians,  and  it  may  perhaps  be  due  to 
the  cutting  off  of  the  supply  of  endotoxins  brought  about  by  the 
ingestion  by  the  leucocytes  of  the  pneumococci  after  opsonization 
by  the  added  serum. 

The  results  are  sufficiently  encouraging  to  lead  us  to  hope  that 
the  serum  may  become  of  more  value  in  the  future,  possibly  as  a 
result  of  the  discovery  of  a  method  by  which  the  dosage  and 
spacing  of  the  injections  and  the  time  for  bleeding  may  be  more 
scientifically  determined. 

In  general,  the  use  of  serum  for  localized  lesions  does  not 
appear  to  be  of  much  value  ;  but  great  benefit  has  been  claimed  to 
follow  the  use  of  Romer's  serum  in  ulcus  serpens  of  the  cornea, 
used  either  alone  or,  as  Axenfeld  recommends,  in  conjunction 
with  a  vaccine.  The  serum  is  to  be  dropped  into  the  conjunctiva 
or  injected  beneath  it. 

Vaccines  have  also  been  used  in  pneumonia  and  in  pneumo- 
coccal  septicaemia  with  good  results.  Thus  Eyre  has  reported 
a  case  of  the  latter  disease  in  which  there  were  metastases 
(purulent)  in  the  subcutaneous  tissues,  the  hip,  the  gluteal 
muscle,  etc.,  and  which  recovered  after  five  injections.  In  another 
case  Eyre  notes,  what  is  of  great  importance,  that  no  benefit 
whatever  was  derived  from  a  stock  vaccine,  and  it  was  only  when 


PRACTICAL   APPLICATIONS  367 

the  patient's  own  culture  was  employed  that  improvement  began. 
It  is  in  general  advisable  to  treat  the  case  with  a  stock  vaccine, 
and  to  commence  to  prepare  a  special  one  if  this  should  fail.  In 
prolonged  cases  it  is  sometimes  of  benefit  to  prepare  a  fresh 
vaccine  after  a  time,  in  order  to  counteract  any  possible  change  of 
type  of  the  organism  in  the  body. 

As  a  rule,  the  first  dose  should  not  exceed  50  millions  for  an 
adult  and  10  millions  for  an  infant,  and,  if  the  disease  is  febrile 
and  the  symptoms  acute,  may  be  decidedly  less.  Subsequent 
doses  may  be  larger,  but  as  a  rule  it  is  not  necessary  to  exceed 
200  or  250  millions.  Probably  the  opsonic  control  is  of  more 
value  here  than  in  most  diseases,  but  in  the  absence  of  this  doses 
may  be  given  every  week  or  ten  days.  The  opsonic  control,  it 
may  be  noted,  is  of  more  value  in  acute  cases  than  in  chronic 
ones,  since  in  the  latter  the  index  may  be  persistently  normal  or 
high,  and  yet  the  disease  yields  readily  to  treatment.  Thus  a  case 
of  pneumococcic  empyema  of  the  frontal  sinus  of  four  years' 
duration,  which  had  been  twice  submitted  to  operation,  had  an 
index  well  above  normal,  and  was  completely  and  permanently 
cured  by  four  injections  of  a  homologous  vaccine  at  intervals  of  a 
fortnight.  As  a  general  rule,  for  the  treatment  of  a  small  lesion 
in  an  adult,  such  as  an  unhealed  sinus  from  an  empyema,  the 
injections  may  be  25,  50,  100,  and  200  millions,  with  an  interval 
of  a  week  between  the  first  two  and  ten  days  between  the 
remainder,  of  course  subject  to  any  indications  which  may  be 
derived  from  clinical  observation.  It  need  scarcely  be  pointed 
out  that  but  little  benefit  can  be  expected  in  cases  in  which  there 
is  a  mechanical  obstacle  to  the  escape  of  pus  or  the  closure  of  an 
abscess.  A  case  of  very  chronic  pulmonary  abscess,  due  to 
pneumococci,  in  which  the  X  rays  showed  a  considerable  amount 
of  thickening,  derived  no  benefit  from  a  long  and  careful  course 
of  vaccine  treatment,  with  and  without  opsonic  control. 

Vaccine  treatment  has  been  used  in  acute  pneumonia  (according 
to  Allen,  routine  injections  of  25  millions  may  be  given),  and  is  of 
especial  value  in  unresolved  consolidation,  which  often  clears  up 
after  its  use  in  a  most  satisfactory  manner,  the  moist  sounds 
clearing  up  in  a  very  short  time,  and  the  general  condition 
improving  rapidly. 

Space  forbids  mention  of  all  the  conditions  in  which  the 
pneumococcus,  perhaps  the  most  protean  of  all  bacteria  in  its 
pathogenic  effects,  has  been  combated  by  vaccine  -  therapy. 


368  GONOCOCCIC    INFECTIONS 

Special  mention  should,  however,  be  made  of  such  serious  con- 
ditions as  ulcus  serpens  and  other  diseases  of  the  eye,  in  which 
good  results  have  -been  obtained  by  Allen  and  others.  In  general, 
all  localized  pneumococcic  diseases  should  be  subjected  to  vaccine 
treatment,  if  not  easily  amenable  to  surgical  operation. 

Gonococcic  Infections. 

Here  again  we  have  to  deal  with  an  organism  which  exerts  its 
pathogenic  effects  mainly  or  entirely  by  means  of  an  endotoxin. 
The  lower  animals  are  entirely  refractory  to  infection  with  living 
cultures,  whereas  the  dead  bodies  of  the  cocci  produce  local 
inflammatory  changes  (peritonitis,  etc.,  according  to  the  region 
into  which  the  inoculation  is  made),  or  death  if  the  dose  is  suffi- 
ciently large.  The  toxicity,  however,  is  but  slight,  and  that  of 
the  filtrate  from  the  cultures  is  extremely  small.  Several 
observers  claim  to  have  produced  a  soluble  exotoxin,  but  there  is 
considerable  doubt  as  to  the  interpretation  of  their  results,  and  in 
all  probability  the  substances  present  in  these  fluids  are  simply 
traces  of  endotoxin,  or  free  receptors  let  loose  by  the  autolysis  of 
a  few  of  the  cocci.  Analogy  with  other  diseases  would  rather 
suggest  the  absence  of  a  powerful  exotoxin.  In  the  great  majority 
of  cases  the  disease  is  a  purely  local  one,  and  the  symptoms  of  a 
general  intoxication  very  slight.  The  endotoxin  is  said  to  be  very 
stable,  resisting  the  temperature  of  boiling  water  for  some  hours. 

The  hgemic  indications  of  immunity  are  but  slight.  Bacterio- 
lysis has  not  been  demonstrated,1  but  there  is  some  evidence  to 
think  that  immunization  may  be  due — at  least,  in  some  cases — to  a 
rise  in  the  opsonic  index,  and  since  some  part  of  the  newly-formed 
opsonin  is,  or  may  be,  thermostable,  it  is  possible  that  some  small 
amount  of  immune  body  may  be  produced.  According  to  Allen, 
the  opsonic  index  in  acute  gonorrhceal  infections  is  somewhat 
below  normal — 0-6  or  07;  and  when  spontaneous  cure  takes  place 
it  rises  gradually,  going  as  high  as  1-6.  If,  however,  it  remains 
subnormal,  the  case  passes  on  into  one  of  intractable  gleet.  But 
in  acute  gonorrhceal  conjunctivitis  in  adults  the  index  may  be  as 
high  as  2-5,  showing,  as  has  been  already  seen  in  other  diseases, 
that  a  high  index  is  no  proof  of  immunity.  Agglutinins  also  occur 
in  the  serum  of  immunized  animals  (Torrey  treated  a  rabbit  the 

1  Torrey  has  recently  shown  that  an  antigonococcic  serum  which  he  has 
prepared  possesses  powerful  bactericidal  properties. 


PRACTICAL  APPLICATIONS  369 

serum  of  which  clumped  at  a  dilution  of  i  :  700,000),  but  this  does 
not  occur  in  man,  and  the  reaction  is  useless  in  diagnosis.  An 
interesting  point  brought  out  by  Torrey's  studies  is  that  gonococcic 
cultures  exhibit  well-marked  differences  inter  se.  The  serum  of  the 
rabbit  alluded  to  above,  which  clumped  its  homologous  culture  at 
i  :  700,000,  clumped  another  culture  only  when  diluted  fifty  times 
or  less.  This  is  of  importance  in  connection  with  the  prepara- 
tions of  vaccines  and  sera.  Torrey  also  showed  that  antigono- 
coccic  serum  contained  a  bacterio-precipitin. 

Clinical  facts  would  suggest  that  the  immunity  to  the  gonococcus 
is  of  very  peculiar  nature.  Thus,  as  Ricketts  well  points  out, 
recovery  is  not  due  to  a  loss  of  virulence  of  the  cocci,  for  they 
remain  potent  to  produce  infection  during  all  stages  of  the  disease. 
Nor  is  it  due  to  the  production  of  acquired  local  immunity,  unless, 
indeed,  this  is  of  such  a  nature  that  it  can  be  very  easily  broken 
down ;  for  the  patient  can  be  reinfected  immediately  after  an 
attack,  or  whilst  the  disease  is  in  a  chronic  stage  in  some  part  of 
the  urethra  or  its  diverticula.  It  is  conceivable  that  the  gono- 
coccus is  very  easily  modified  by  passage  through  other  human 
beings,  and  so  altered  that  it  is  able  to  reinfect  a  person  who  has 
just  recovered  from  an  attack  caused  by  it  in  its  unaltered  form ; 
but  this  would  hardly  explain  the  recrudescence  of  an  apparently 
cured  discharge  after  excessive  indulgence  in  alcohol.  Even  the 
relationship  between  phagocytosis  and  recovery  is  not  easy  to 
make  out.  Unlike  other  organisms,  the  gonococci  do  not  appear 
to  undergo  the  usual  morphological  changes  indicative  of  intra- 
cellular  digestion  (loss  of  staining  power  and  of  sharp  outline) 
usually  seen  in  bacteria  after  phagocytosis ;  nor  do  the  leucocytes 
which  have  taken  them  up  show  degenerative  changes  (Ricketts). 
And  the  fact  that  the  gonococci  are  practically  all  intracellular 
from  a  very  early  period  in  the  disease — indeed,  whilst  it  is  in 
active  progress — would  seem  to  indicate  that  it  is  a  most  inefficient 
protective  mechanism.  Further,  there  is  no  obvious  difference 
in  the  phagocytosis  according  to  the  height  of  the  opsonic 
index,  which  would  lead  us  to  believe  that,  even  when  the  index 
is  low,  there  is  sufficient  opsonin  to  enable  all  the  available  cocci 
to  be  taken  up.  As  far  as  it  goes,  the  evidence  leads  us  .to  think 
that  the  process  of  cure  is  due  to  some  local  change — possibly  to 
some  exhaustion  of  a  necessary  nutrient  material — and  not  to  a 
general  haemic  reaction. 

Natural  local  immunity  is  very  marked  in  the  case  of  this 

24 


370  GONOCOCCIC  INFECTIONS 

organism.  A  spread  of  the  disease  beyond  the  mucous  membrane 
of  the  urethra  is  extremely  rare.  Less  rare,  though  still  un- 
common, is  extension  to  the  bladder.  Haemic  infections  are 
also  rare,  but  do  occur ;  in  them,  as  in  other  general  infections,  it 
is  difficult  to  see  how  the  cocci  can  run  the  gauntlet  of  the  leuco- 
cytes. 

Diagnosis. — In  most  cases,  of  course,  this  is  made  by  the 
demonstration  of  the  specific  coccus— usually  an  easy  task.  If  no 
material  is  forthcoming  (as  in  gonorrhceal  arthritis,  etc.),  two 
methods  are  available — the  opsonic  index  and  the  absorption  of 
complement.  According  to  Allen,  the  normal  index  ranges 
between  0-8  and  1-2,  and  in  chronic  infections  it  may  be  low 
(down  to  o!2)  or  high  (up  to  2  or  more).  Further,  a  dose  of 
75  millions  of  dead  cocci  causes  but  little  disturbance  in  a  non- 
gonorrhoeal  patient,  but  a  marked  rise  if  the  patient  is  infected. 
The  method  of  absorption  of  complement  has  been  employed 
with  marked  success  by  Meakins,  and  appears  to  be  of  great 
value.  The  difficulty  of  the  technique,  however,  renders  it  much 
less  easily  available  than  the  opsonic  method. 

Treatment. — The  use  of  serum  need  hardly  be  discussed.  Some 
cases,  it  is  true,  have  apparently  been  benefited,  but  it  is  always 
possible  that  the  serum  may  have  contained  specific  toxic  bodies 
which  acted  as  a  vaccine. 

The  vaccine  treatment,  on  the  other  hand,  is  of  the  greatest 
possible  value.  The  dose  varies  between  5  and  500  millions. 
In  general,  large  amounts  are  not  well  tolerated,  especially  early 
in  the  treatment.  The  doses  may,  of  course,  be  regulated  by  the 
opsonic  index,  but  this  is  probably  not  necessary.  In  acute  cases 
two  or  three  doses  of  50  millions  may  be  given  ;  but  it  is  in  the 
chronic  infections,  especially,  perhaps,  in  gonorrhceal  arthritis, 
that  the  method  is  of  especial  value.  Here  the  dose  may  begin 
with  50  millions,  rising  to  500  millions,  or  even  twice  this  amount, 
and  the  intervals  may  be  seven  to  ten  days.  If  no  benefit  is 
obtained  a  further  course  of  treatment  under  opsonic  control  may 
be  administered.  The  results  are  usually  beneficial  in  the 
extreme. 

Gonorrhceal  conjunctivitis  is  to  be  treated  on  the  same  lines  as 
acute  arthritis,  but  here,  of  course,  ordinary  local  antiseptic 
treatment  is  all-important.  Gonorrhceal  iritis  is  treated  like 
gonorrhceal  arthritis,  and  the  results  are  usually  excellent. 


PRACTICAL   APPLICATIONS  371 

Meningococcic  Infections. 

Very  little  is  known  concerning  the  toxin  of  the  meningococcus. 
Cultures  are  of  very  feeble  toxicity  for  animals,  and  large  doses 
of  the  vaccine  are  usually  (though  not  invariably)  well  tolerated 
by  human  patients.  It  is  probably  an  endotoxin  which  is  only 
produced  under  certain  conditions.  Its  effect  in  the  human 
subject  is  mainly  a  local  one,  manifested  chiefly  on  the  tissues  in 
or  near  which  the  cocci  are  localized.  The  disease  is  in  most 
cases  a  local  one,  the  organism  being  rarely  found  in  the  blood 
or  organs  other  than  the  brain  and  cord. 

The  frequency  with  which  the  organism  is  found  within  the 
polynuclear  leucocytes  would  lead  us  to  believe  that  the  organism 
is  combated  in  the  main  by  phagocytosis  ;  and  this  is  confirmed 
on  the  whole  by  the  results  obtained  by  a  study  of  the  disease  by 
Wright's  method.  When  opsonic  determinations  are  made  using 
a  virulent  culture,  such  as  one  derived  from  the  patient  himself, 
it  is  found  as  a  rule  to  be  taken  up  very  badly  in  preparations 
made  with  normal  serum  and  leucocytes,  and  very  well  when 
some  of  the  patient's  serum  is  present,  so  that  very  high  indices 
are  obtained.  This  is  obviously  because  the  cocci  have  become 
animalized,  like  the  virulent  pneumococci  studied  by  Rosenow 
and  others.  They  require  a  large  dose  of  opsonin,  such  as  is 
present  in  serum  from  a  patient  who  is  attempting  to  combat  the 
disease,  and  not  in  that  from  a  normal  person.  The  result  is 
that  the  opsonic  index  of  these  patients  is  often  extremely  high, 
figures  of  10  or  more  being  common,  and  40  has  been  recorded. 
Houston  has  pointed  out  that  old  laboratory  cultures  which  have 
lost  their  virulence  are  phagocyted  more  easily  in  presence  of 
normal  serum,  so  that  the  opsonic  index  of  meningitis  patients, 
as  determined  by  the  use  of  these  non-virulent  cultures,  is  com- 
paratively low.  Houston  has  proposed  this  test  for  distinguishing 
between  the  "  true "  meningococcus  and  allied  cocci  of  similar 
morphological  characters.  He  determines  the  opsonic  index  of  a 
patient  suffering  from  true  cerebro-spinal  meningitis  against  a 
normal  control,  using  both  a  known  meningococcus  culture  and 
that  under  consideration.  In  what  he  terms  the  positive  reaction 
there  is,  in  the  case  of  the  normal  blood,  very  little  phagocytosis, 
and  no  agglutination  of  the  cocci  which  are  not  ingested,  whilst 
with  the  blood  from  the  cerebro-spinal  case  there  is  much  phago- 
cytosis and  marked  clumping  of  the  free  cocci.  It  would  seem, 

24—2 


372  MENINGOCOCCIC    INFECTIONS 

however,  that  the  test  is  simply  one  of  the  virulence  of  the 
culture,  rather  than  that  of  its  specific  nature.  As  regards 
agglutination,  Houston  and  Rankin  point  out  that  this  property 
also  is  lost  on  prolonged  cultivation  on  artificial  media ;  and  as 
regards  the  behaviour  of  the  coccus  in  opsonic  estimations,  exactly 
the  same  phenomena  are  seen  when  cultures  isolated  from  what 
are  apparently  ordinary  cases  of  basic  meningitis  are  tested  against 
the  blood  of  the  patient  and  a  normal  control.  It  seems  more 
reasonable  to  suppose  that  the  coccus  from  the  basic  meningitis 
cases  are  either  of  lower  virulence,  or  that  their  virulence  has 
developed  along  different  lines  from  a  common  non-virulent 
stock,  rather  than  a  different  species. 

The  opsonin  present  in  the  serum  in  meningitis  is  to  a  large 
extent  thermolabile.  Some  thermostable  opsonin  is  present,  the 
amount  being  roughly  proportionate  to  the  height  of  the  index. 

Agglutination  is  somewhat  difficult  of  study  in  the  case  of  this 
organism,  owing  to  the  variations  presented  by  different  cultures 
in  this  respect  and  the  difficulty  in  procuring  a  homogeneous 
emulsion.  Tested  by  ordinary  methods,  the  blood  of  meningitis 
cases  does  not  clump  in  high  dilutions  :  according  to  Davis,  i  :  50 
is  about  the  average.  Kutscher  states  that  the  phenomenon  is 
much  more  marked  at  55°  C.,  and  that  a  culture  which  was  not 
agglutinated  at  all  by  a  specific  serum  at  37°  C.  was  clumped  in 
twenty-four  hours  at  55°  C.  in  dilutions  of  i  :  500,  or  even  i  :  1,000. 
If  this  is  correct  it  may  prove  important  in  the  clinical  diagnosis 
of  the  disease,  which  at  present  is  based  mainly  on  the  characters 
of  the  cerebro-spinal  fluid,  and  especially  on  the  presence  of  the 
coccus.  The  cytological  and  chemical  examination  of  the  fluid 
affords  definite  evidence  of  the  presence  of  a  meningitis,  but  the 
cocci  are  not  always  discoverable,  especially  in  cases  of  internal 
meningitis,  or  those  in  which  the  foramina  at  the  base  of  the  brain 
are  closed.  The  opsonic  index  may  be  of  great  value,  especially 
if  a  virulent  culture  is  at  hand.  According  to  Houston  and 
Rankin,  the  positive  "  reaction  "  described  above  is  not  usually 
present  before  the  fifth  or  sixth  day  in  the  epidemic  form. 

The  serum  of  immunized  animals  contains  a  substance  which 
gives  the  phenomenon  of  fixation  of  complement  when  com- 
bined with  meningococci,  and  this  fact  is  used  by  Kolle  in  the 
standardization  of  his  therapeutic  serum ;  but  whether  this  is  a 
bacteriolysin  or  bactericidal  substance  is  not  definitely  known. 
A  cording  to  Davis,  normal  blood-serum  is  bactericidal  to  menin- 


PRACTICAL   APPLICATIONS  373 

gococci,  and  this  power  is  increased  in  meningitis.  He  states, 
however,  that  the  opsonic  power  of  the  blood  was  not  altered 
during  the  disease. 

The  treatment  differs  in  the  acute  and  chronic  stages.  In  the 
very  acute  cases  the  only  hope  appears  to  be  in  the  use  of 
a  serum,  coupled  of  course  with  the  more  ordinary  remedial 
measures,  such  as  repeated  lumbar  puncture.  This  probably  acts  by 
removing  the  inert  cerebro-spinal  fluid  (which  is  almost  deficient 
both  in  opsonin  and  in  complement),  and  causing  the  exudation 
of  fluid  containing  a  larger  amount  of  protective  substances.  In 
more  chronic  cases,  and  especially  in  posterior  basic  meningitis, 
the  use  of  vaccines,  either  alone  or  in  conjunction  with  serum,  is 
more  promising. 

Several  sera  have  been  prepared — Kolle  and  Wassermann's, 
Jochmann's,  Ruppel's,  Flexner's,and  Burroughs  and  Wellcome's — 
and  very  diverse  reports  have  been  published  concerning  their 
value.  Small  differences  exist  in  the  methods  of  preparation, 
but  in  all  cases  large  doses  of  organisms,  dead  or  living,  are 
injected,  either  subcutaneously  or  into  the  veins.  The  potency  of 
the  serum  is-  tested  either  by  agglutination  or  by  the  absorption  of 
complement.  According  to  Houston  and  Rankin,  some  of  the 
therapeutic  sera  in  common  use  have  very  slight  opsonic 
power  and  are  devoid  of  agglutinating  properties.  It  seems  quite 
clear  that  the  serum  is  useless  if  given  hypodermically,  and  the 
observers  who  have  obtained  beneficial  results  (Flexner,  Levy, 
and  others)  have  injected  the  serum  into  the  spinal  canal  after 
removing  an  equal  amount  of  cerebro-spinal  fluid  by  lumbar 
puncture.  The  dose  is  20  to  30  c.c.,  repeated  several  times  at 
intervals  of  twenty-four  hours.  Some  patients  (adults)  have 
received  as  much  as  340  c.c.  The  injections  may  cause  vomiting 
and  unconsciousness,  but  no  permanent  inconvenience  ;  they  are 
painful,  and  this  is  attributed  to  the  use  of  carbolic  acid  as  a 
preservative  agent.  The  treatment  must  be  commenced  early, 
and  in  view  of  the  results  of  Levy,  in  whose  practice  the  deaths  fell 
from  78-57  per  cent,  to  6-25  per  cent.,  and  Flexner,  who  reduced 
the  mortality  to  20  per  cent.,  seems  to  be  of  decided  value.  Other 
observers  have,  however,  been  less  successful. 

The  use  of  serum  from  convalescent  cases  of  meningitis  (which 
they  find  to  possess  marked  bactericidal  properties)  has  been 
suggested  by  MacKenzie  and  Martin.  The  blood  is  collected  by 
venesection,  whipped,  and  centrifugalized,  and  the  serum  used 


374  MALTA    FEVER 

for  intraspinous  injection.  In  some  cases  the  blood  used  was 
taken  from  the  patient  himself,  and  they  obtained  encouraging 
results  after  the  use  of  both  methods.  The  method  is  perfectly 
rational,  and  we  might  expect  on  a  priori  grounds  that  fresh  serum, 
containing  its  full  amount  of  complement  and  thermolabile  opsonin, 
would  be  of  more  benefit  than  stale  immune  serum.  With  the 
idea  of  causing  an  increased  outflow  of  preventive  substances 
from  the  patient's  blood  by  producing  a  mild  aseptic  inflammatory 
process,  Briscoe  has  injected  dilute  carbolic  acid  solutions  and 
other  fluids  into  the  cerebro-spinal  canal,  but  without  success. 

The  treatment  by  vaccines  has  not  attracted  much  attention, 
but  in  the  subacute  and  chronic  cases  it  is  well  worthy  of  a  trial. 
I  have  used  it  in  four  cases,  with  three  recoveries  and  one  death. 
These  figures  are  not  sufficiently  great  to  argue  about,  but  what 
was  most  obvious  was  the  very  decided  clinical  improvement 
which  followed  almost  every  dose.  This  occurred  too  frequently 
to  be  mere  coincidences,  and  as  these  patients  were  all  young 
children  the  question  of  their  being  due  to  a  mental  effect  need 
not  be  considered.  The  dose  has  been  250  millions,  increasing  to 
500  millions,  and  1,000  millions,  and  no  bad  effects  have  been 
noticed.  They  have  been  usually  regulated  by  opsonic  control, 
and  the  improvement  in  the  general  condition  as  the  index  rises 
appears  to  me  to  be  more  definite  in  this  disease  than  in  most 
others.  It  need  hardly  be  pointed  out  that  no  form  of  specific 
treatment  will  cure  the  obliteration  of  the  foramina  in  the  roof  of 
the  fourth  ventricle  and  consequent  hydrocephalus,  which  is  so 
frequently  present  in  these  chronic  cases. 

In  some  cases  the  index  shows  a  very  marked  rise  (to  10  or 
more)  as  a  result  of  a  single  injection  of  a  homologous  vaccine, 
whilst  in  others  the  reaction  is  much  less,  and  the  level  does  not 
rise  much  above  unity.  It  is  probable  that  these  two  types  of 
reaction  correspond  to  infections  with  the  cerebro-spinal  and  basic 
meningitis  types  of  organism,  but  the  cases  show  no  obvious 
clinical  difference. 

Malta  Fever. 

The  toxin  of  Malta  fever  appears  to  be  an  endotoxin.  Killed 
cultures  are  decidedly  toxic  for  animals,  and  their  prophylactic 
use  in  moderate  doses  has  been  followed  in  some  cases  by  the 
development  of  chronic  aseptic  abscesses  (Eyre,  Bousfield). 
The  type  of  immunity  is  not  known  ;  no  bactericidal  substance  is 


PRACTICAL   APPLICATIONS  375 

known  to  be  developed  in  the  blood,  and  Eyre's  attempts  to 
produce  a  powerful  curative  or  preventive  serum  were  unsuccessful. 
There  is  more  evidence  pointing  to  opsonic  action  in  the  cure  of 
the  disease  and  the  subsequent  immunity :  the  index  early  in  the 
disease  is  as  a  rule  low,  and  it  rises  during  the  progress  of  the 
case.  In  some  points  the  immunity  reactions  of  the  disease 
resemble  tubercle  (slow  development  of  the  immunity,  frequency 
of  relapses,  absence  of  known  bacteriolytic  properties  in  the 
serum,  local  toxicity  of  the  cultures),  but  there  is  this  marked 
difference,  that  Malta  fever  is  essentially  a  septicaemia,  the  cocci 
being  present  in  the  blood  in  small  or  large  numbers  in  practically 
all  cases. 

The  agglutinative  reaction,  first  studied  by  Wright  and  Smith 
is  of  enormous  importance  in  the  diagnosis  of  the  disease,  and 
it  was  Zammit's  discovery  of  clumping  powers  in  the  blood  of 
the  goat  that  led  to  the  discovery  of  the  fact  that  the  milk  of 
these  animals  was  frequently  infective  even  when  the  animal  had 
no  signs  of  disease.  Remedial  measures  based  on  this  phenomenon 
have  been  of  enormous  advantage  in  checking  the  disease. 

Various  criteria  are  adopted  in  the  application  of  the  reaction 
to  diagnosis  of  disease  in  man.  Critien  uses  a  dilution  of  i  :  10, 
and  a  time-limit  of  half  an  hour,  and  finds  results  thus  obtained 
as  conclusive  as  if  higher  dilutions  were  used.  Bassett-Smith 
recommends  i  :  30,  with  a  time-limit  of  four  hours,  or  twenty- 
four  if  the  macroscopic  method  be  used.  Eyre,  however,  points 
out  that  the  blood  not  infrequently  clumps  in  a  high  dilution,  but 
not  when  more  concentrated — e.g.,  giving  no  reaction  between 
i  :  10  and  i  :  100,  but  clumping  strongly  at  i  :  200.  It 
appears,  therefore,  that  a  series  of  dilutions  should  be  made  if 
negative  results  are  obtained  with  the  test  adopted  as  a  standard. 
Agglutination  may  be  manifested  in  very  high  dilutions,  even  as 
high  as  i  :  500,000.  It  usually  appears  about  the  fifth  day. 
The  only  other  certain  method  of  diagnosis  is  the  isolation  of  the 
organism  from  the  blood,  usually  a  fairly  easy  matter. 

The  treatment  of  the  disease  by  means  of  vaccines  has  been 
studied  by  Reid,  and  more  recently  by  Bassett-Smith.  The 
latter  recommends  a  ten-day-old  agar  culture,  emulsified  in  dis- 
tilled water,  sterilized  at  60°  C.  for  an  hour,  and  sufficient  carbolic 
acid  added  to  bring  the  strength  to  0-5  per  cent.  It  is  standardized 
by  drying  20  c.c.  (of  course,  before  the  addition  of  the  carbolic 
acid),  and  weighing  the  solid  residue.  The  results  thus  obtained 


376  TUBERCULOSIS 

are  used  to  determine  the  degree  of  dilution  necessary,  so  that 
20  c.c.  of  the  vaccine  should  contain  4  milligrammes  of  solid 
substance.  The  doses  given  were  between  0*25  and  0*5  c.c.  They 
were  checked  by  opsonic  control  twice  weekly,  and  were  in 
general  given  once  a  week  or  fortnight.  Bassett-Smith  does  not 
approve  of  the  method  in  acute  cases,  but  his  chronic  ones 
seemed  decidedly  benefited. 

The  prophylactic  use  of  the  vaccine  has  not  been  tried  on  a 
scale  sufficiently  large  to  enable  any  very  definite  inferences  to 
be  drawn  therefrom.  The  doses  may  be  200  to  400  million 
cocci,  and  in  general  two  are  given,  the  results  being  judged  by 
their  effect  on  the  agglutination  reaction.  As  noted  above,  some 
bad  local  results  have  been  noticed,  and  there  is  in  all  cases  a 
good  deal  of  fever,  malaise,  and  inflammation  at  the  site  of  injection 
and  corresponding  lymph  glands. 

Tubercle. 

The  actual  toxin  and  mode  of  action  of  the  tubercle  bacillus 
remain  unknown.  The  most  interesting  substance  which  has 
been  derived  from  cultures  of  the  organism  is  tuberculin,  which 
has  been  discussed  previously.  That  it  is  not  the  true  toxin 
appears  from  the  fact  that  animals  immunized  to  it  are  still 
susceptible  to  the  tubercle  bacillus,  and  a  serum  can  be  prepared 
which  appears  to  be  an  antitoxin  for  tuberculin,  but  it  has  no 
curative  or  preventive  effects  in  tubercle.  The  fatty  substances 
which  confer  on  the  bacillus  its  peculiar  staining  properties  are 
not  devoid  of  toxicity.  They  cause  chronic  inflammatory  and 
caseous  changes  in  the  tissues,  and  may  perhaps  play  a  larger 
role  in  the  evolution  of  the  lesion  than  is  generally  thought.  And 
it  must  be  pointed  out  that  tuberculosis  may  be  an  afebrile 
disease  throughout.  The  temperature  of  phthisis  is  mainly  due  to 
other  organisms,  notably  to  the  pyogenic  organisms  which  form 
such  frequent  secondary  contaminations  of  the  vomicae.  The 
chief  pure  tuberculous  affection  in  which  fever  is  a  marked  and 
constant  symptom  is  tubercle  of  the  meninges,  and  here  the  local 
processes  are  in  close  proximity  to  the  cerebral  cortex.  In  general 
tuberculosis  there  is  usually  fever,  but  there  is  also  usually  a 
focus  exposed  to  secondary  infections,  or  a  broncho-pneumonia  in 
which  other  organisms  play  a  part.  The  main  toxic  effect  which 
we  recognize  as  being  due  to  the  tubercle  bacillus  is  exerted  on 
the  surrounding  tissues,  and  there  is  no  disease  which  is  more 


PRACTICAL  APPLICATIONS  377 

truly  a  local  one  ;  meaning  thereby  that  in  pure  tuberculosis  the 
general  symptoms  indicative  of  a  spread  of  the  toxins  into  the 
general  circulation  are  but  slight,  and  the  local  lesion  constitutes 
almost  the  whole  of  the  disease.  Note,  for  instance,  the  good 
general  health  which  often  occurs  in  patients  with  tuberculous 
glands  in  the  neck,  even  if  of  some  size.  And  where  the  general 
health  is  impaired,  it  will  often  be  found  to  be  a  predisposing 
cause  rather  than  a  result  of  the  tuberculous  process. 

Immunity. — These  facts  would  tend  to  show,  what  I  believe  to 
be  the  case,  that  the  immunity  to  tubercle  is  local  rather  than 
general.  That  general  immunity  exists  there  can  be  no  doubt, 
but  we  must  distinguish  between  general  immunity  due  to  a 
condition  of  the  blood  and  that  due  to  a  resisting  power  inherent 
in  all  the  tissues  of  the  body.  Thus  to  take  two  patients,  one 
strong  and  robust  and  the  other  of  enfeebled  vitality  :  the  latter 
may  fall  a  ready  victim  to  tubercle,  whilst  the  former  resists 
exposure  to  the  most  virulent  infection  :  yet  there  may  be  no 
demonstrable  difference  in  the  serum  of  the  two  persons.  There 
may,  it  is  true,  be  a  slight  difference  in  the  opsonic  index,  but  this 
is  not  invariably  the  case. 

The  importance  of  local  immunity  in  tubercle  appears  clearly 
from  the  fact  that  areas  of  advance  and  of  cure  may  occur  in  the 
same  patient,  or  even  in  different  parts  of  the  same  lesion.  This 
is  often  very  well  seen  in  cases  of  lupus,  but  is  even  more 
marked  in  sections  from  chronic  tuberculosis — e.g.,  of  a  synovial 
membrane,  in  which  areas  of  cure  (as  indicated  by  fibrosis  and 
organization  of  the  giant-cell  systems)  and  of  extension  (as  in- 
dicated by  caseation  and  the  formation  of  new  tubercles)  may 
almost  always  be  found  in  the  same  lesion.  In  general,  tubercle 
tends  to  spontaneous  cure,  and  when  a  lesion  continues  to  spread 
it  will  often  be  found  that  some  second  influence  (such  as  a 
secondary  infection)  is  at  work  ;  even  in  debilitated  subjects  there 
is  usually  an  effort  at  spontaneous  cure  in  some  parts  of  the 
lesion. 

As  regards  the  nature  of  this  process  of  cure,  we  can  say  but 
little.  There  can  be  but  little  doubt  that  the  main  curative 
agency  is  phagocytosis,  but  there  is  no  reason  to  think  that  this 
is  dependent  on  the  same  mechanism  of  opsonic  action  that  we 
reproduce  so  readily  in  vitro.  Polynuclear  leucocytes  are  con- 
spicuous by  their  absence  from  tubercles,  and  the  only  cells  in 
which  tubercle  bacilli  have  actually  been  demonstrated  in  the 


37^  TUBERCULOSIS 

living  body  are  the  giant  and  endothelial  cells.  In  these,  as 
Metchnikoff  pointed  out  many  years  ago,  tubercle  bacilli  can  be 
seen  in  all  stages,  of  degeneration,  from  bacilli  possessed  of  normal 
staining  properties  to  mere  "  ghosts."  Either  the  endothelial 
and  giant  cells  actually  engulf  the  bacilli,  or,  what  is  perhaps  more 
likely,  they  grow  round  them,  being  stimulated  to  growth  and 
proliferation  by  the  action  of  the  same  toxin  which,  when  more 
concentrated,  leads  to  caseation  and  death.  Whether  any 
opsonin  is  necessary  for  this  process  to  occur,  or  whether  this 
opsonin  is  produced  locally  or  makes  its  way  from  the  blood,  we 
do  not  know.  What  we  do  know,  however,  is  that  a  high  opsonic 
index  does  not  necessarily  imply  that  the  local  lesions  are  under- 
going cure  ;  chronic  tubercle,  and  especially  lupus  of  some  stand- 
ing, is  often  associated  with  a  very  high  index.  But  Bulloch 
found  that  the  cases  of  lupus  which  did  well  on  X-ray  treatment 
(which,  amongst  other  effects,  causes  a  permeation  of  the  lesions 
with  plasma)  were  those  that  had  a  high  index,  and  that  in  the 
cases  in  which  it  was  low  originally  better  results  could  be 
obtained  if  it  were  raised  by  means  of  tuberculin  injections.  It 
is,  therefore,  not  improbable  that  opsonin  may  actually  soak 
through  the  lymphoid  zone  and  sensitize  the  bacilli,  thus  aiding 
phagocytosis  by  the  giant  cells.  But  this  is  by  no  means  certain. 
The  results  of  an  injection  of  tuberculin  are  probably  very 
complex,  and  it  is  at  least  possible  that  the  curative  effect  is 
mainly  or  entirely  due  to  the  production  of  a  local  reaction  in  the 
neighbourhood  of  the  lesion,  as  a  result  of  which  it  is  flushed 
with  blood,  and  perhaps  simply  increased  as  regards  nutrition. 
Very  small  doses  of  tuberculin  are  sufficient  to  cause  a  slight  but 
definite  reaction.  This  may  be  quite  inappreciable  in  most 
regions,  but  perfectly  obvious  in  the  case  of  tubercles  of  the  iris, 
which  may  be  seen  to  become  surrounded  by  a  hyperaemic  zone 
after  injections  of  T^-^  milligramme  of  new  .tuberculin,  or  even 
less.  Reactions  of  this  nature  are,  of  course,  entirely  harmless, 
and  are  unaccompanied  byu!he  slightest  rise  of  temperature,  if  any. 
The  only  role  which  we  can  assign  with  any  degree  of  probability 
to  the  polynuclear  leucocyte  in  the  struggle  against  tubercle  has 
regard  to  its  action  on  bacilli  which  have  made  their  way  into  the 
blood.  Here  the  conditions  are  much  more  like  those  which 
occur  in  our  opsonin  experiments,  and  it  is  quite  likely  that 
opsonization  and  phagocytosis  occur.  Even  this  is  not  certain, 
however,  for  we  do  not  yet  know  definitely  whether  opsonin  exists 


PRACTICAL   APPLICATIONS  379 

in  the  circulating  blood-plasma,  and  that  the  mechanism  is  of 
comparatively  little  value  appears  from  the  fact  that  rupture  of  a 
caseous  gland  into  a  vein  is  so  often  (as  far  as  we  know,  always) 
followed  by  general  tuberculosis.  It  is  possible  that  living  and 
virulent  bacilli  may  be  taken  up  by  the  polynuclear  leucocytes, 
carried  to  distant  organs,  such  as  the  lymph  glands  or  spleen,  and 
there  continue  to  grow,  producing  anatomical  tubercles. 

As  regards  the  nature  of  tuberculo-opsonin,  the  normal  opsonin 
is  completely  thermolabile,  and  the  rise  in  the  index  due  to 
injections  or  the  natural  disease  is  mainly  of  that  nature  also. 
Occasionally  the  presence  of  thermostable  opsonin  is  demonstrable, 
but  it  never  becomes  abundant. 

We  know  but  little  concerning  any  other  antibody  or  defensive 
mechanism.  No  bacteriolytic  or  bactericidal  action  is  demon- 
strable. This  may  possibly  be  due  to  the  technical  difficulties 
incidental  to  investigations  of  this  nature  on  a  bacillus  which 
owes  its  staining  properties  to  the  presence  of  fats  (which  we 
cannot  expect  a  serum  to  dissolve),  and  which  grows  so  slowly. 
Wassermann  and  others  have  shown  that  antibodies  occur  in 
patients  immunized  with  tuberculin,  but  we  do  not  know  their 
nature.  Nothing  really  definite  is  known  concerning  an  antitoxin. 
An  agglutinin  is  often  formed,  but  it  may  be  absent  throughout 
the  whole  course  of  the  disease,  so  that  we  cannot  regard  it  as  of 
importance. 

Diagnosis. — Where  possible  this  should  be  made  by  the  recogni- 
tion of  the  bacillus,  an  achievement  which  modern  methods 
have  made  possible  in  many  cases  in  which  it  would  formerly 
have  been  regarded  as  out  of  the  question.  Into  this  and  into 
questions  of  cytology,  etc.,  we  need  not  enter.  The  main  methods 
to  be  considered  are — (i)  the  tuberculin  reaction,  and  (2)  the 
opsonic  index. 

i.  The  Tuberculin  Reaction. — The  old  tuberculin  is  used,  and  is  best 
bought  ready  prepared,  as  issued  with  the  German  Government 
stamp.  It  may  be  standardized,  but  this  process  is  uncertain, 
animals  differing  markedly  in  susceptibility.  Several  methods 
have  been  introduced,  but  are  not  in  general  use.  Behring's 
method  is  to  determine  the  lethal  dose  for  guinea-pigs  on  sub- 
cutaneous injection,  whilst  Von  Lingelsheim  injects  directly  into 
the  brain,  the  susceptibility  of  which  is  much  greater.  As  a  rule, 
a  normal  guinea-pig  will  stand  a  dose  of  i  c.c.  with  impunity.  It 
should  be  diluted  before  use  with  sterile  normal  saline  solution 


380  TUBERCULOSIS 

containing  0-5  per  cent,  carbolic  acid  or  lysol,  and  kept  in  sealed 
"  ampoules,"  each  containing  i  c.c.  A  convenient  method  to 
adopt  is  to  take  99  c.c.  of  diluent  and  add  the  whole  of  a  i  c.c. 
bottle  of  tuberculin.  Each  cubic  centimetre  of  this  contains  10  milli- 
grammes of  fluid,  or  yj^  c.c. ;  several  ampoules  are  prepared,  and  the 
remainder  of  the  fluid  diluted  with  an  equal  amount  of  the  diluent. 
Each  cubic  centimetre  now  contains  5  milligrammes,  or  -^^  c.c.,  and 
several  ampoules  are  filled  with  this  dilution.  Lastly,  what  remains, 
or  part  of  it,  is  diluted  with  four  times  its  volume  of  diluent,  so  that 
each  cubic  centimetre  contains  i  milligramme,  or  y^^-  c.c.,  of  tuber- 
culin. It  will  be  noted  that  the  fraction  of  a  milligramme  refers  to  the 
tuberculin  as  sold,  and  not  to  any  active  constituent  it  is  supposed  to 
contain.  It  is  recommended  to  err  on  the  side  of  caution,  and  to  com- 
mence with  y^1^  c.c.,  and  then  to  go  on  to  ^J^  and  finally  y^-;  but 
with  proper  selection  of  cases  it  is  probable  that  the  risk  with  ^ J^  is 
infinitesimal.  The  rise  in  temperature  which  constitutes  a  reaction 
should  be  at  least  i°  F.,  and  is  usually  more.  It  usually  com- 
mences in  eight  to  twelve  hours,  rises  for  another  two  hours, 
remains  up  for  a  few  hours  longer,  and  then  falls  rapidly.  There 
is  often  a  considerable  amount  of  general  malaise. 

It  should  be  used  only  in  cases  in  which  the  diagnosis  cannot 
be  made  by  other  methods  with  ease  and  certainty,  and  in  which 
it  is  of  great  importance  that  it  should  be  made  definitely  and 
rapidly.  Thus,  a  patient  living  under  favourable  conditions  who 
developed  ambiguous  indications  of  phthisis  might  fairly  be 
watched  for  a  time,  his  weight  recorded,  an  attempt  made  to 
obtain  sputum,  etc.  But  the  same  signs  occurring  in  a  person 
who  was  about  to  get  married,  or  a  medical  man  thinking  of 
taking  a  resident  appointment  in  a  hospital,  would  be  a  strong 
indication  for  the  diagnostic  use  of  tuberculin.  Perhaps  its  main 
value  is  that  it  enables  a  negative  diagnosis  to  be  made  with  some 
degree  of  confidence,  and  is  the  only  agent  which  will  do  so.  No 
reaction  after  two  injections  of  yj^  c.c.  may  be  taken  as  definite 
proof  that  the  disease  is  absent. 

It  should  not  be  used  (i)  when  the  temperature  is  irregular — 
obviously,  since  the  rise  might  not  be  due  to  the  tuberculin — and 
(2)  when  there  are  secondary  infections. 

Some  cases  of  syphilis,  leprosy,  and  actinomycosis  are  said  to 
react,  but  this  is  unusual,  and  the  concomitant  presence  of  tubercle 
has  not  always  been  ruled  out. 

In  cattle  the  doses  are  larger.     The  tuberculin  is  usually  diluted 


PRACTICAL  APPLICATIONS  381 

ten  times,  and  the  dose  varies  from  4  c.c.  for  a  large  bull  to  i  c.c. 
for  a  calf  under  one  year.  The  temperature  is  taken  for  a  day  or 
two  before  the  injection,  and  at  the  ninth,  twelfth,  fifteenth,  and 
eighteenth  hours  afterwards.  The  rise  is  gradual,  reaching  its 
maximum  in  twelve  to  fifteen  hours,  and  then  falling  gradually  to 
normal.  According  to  Nocard,  anything  over  2*5°  F.  is  diagnostic, 
from  i -4°  to  2-5°  F.  suspicious,  and  under  i'4°  F.  unimportant.  The 
temperature  of  the  animal  should  not  exceed  103°  F.  when  the 
injection  is  made. 

Von  Pirquefs  reaction  or  the  cutireaction  :  This  was  introduced 
as  a  method  of  avoiding  the  dangers  supposed  to  be  incidental  to 
the  use  of  tuberculin  sub  cute.  A  very  gentle  scarification  of  the 
skin  is  made,  just  as  for  Jennerian  vaccination,  avoiding  drawing 
blood,  if  possible,  and  the  abraded  surface  covered  with  diluted 
tuberculin  (i  part  with  3  of  normal  saline  containing  0-25  per  cent, 
carbolic  acid).  A  control  scarification  is  made,  preferably  on  the 
other  arm,  and  covered  with  the  carbolized  normal  saline  solution. 
The  reaction  takes  the  form  of  a  red  papule  of  varying  size, 
sometimes  extending  for  ^  inch  or  more  in  all  directions  from  the 
site  of  the  original  scarification.  I  have  seen  the  skin  so  sensitive 
that  a  vivid  reaction  was  obtained  where  the  diluted  tuberculin 
had  accidentally  run  down  the  skin.  The  redness  increases  for  a 
day  or  two,  and  may  remain  for  four  or  five  days,  but  in  general 
disappears  earlier.  It  is  to  be  compared  with  the  control  side, 
which  has  also  been  treated  with  dilute  carbolic  acid,  but  no 
tuberculin.  Von  Pirquet  now  uses  undiluted  tuberculin,  the 
control  scarification  not  being  treated  at  all.  This  is  probably 
the  better  method,  and  seems  devoid  of  danger.  In  Moro's 
method  an  ointment  containing  tuberculin  is  rubbed  into  the  skin. 

The  process  is  probably  quite  devoid  of  danger.  Some  tuber- 
culin is  certainly  absorbed,  since  I  have  seen  a  quite  definite  local 
reaction  round  a  tubercle  in  the  iris;  the  temperature,  however, 
does  not  usually  rise,  but  may  do  so ;  this  is  a  sign  that  the 
scarification  has  been  too  deep.  No  reaction  is  given  in  advanced 
cases  of  the  disease,  probably  because  the  resisting  powers  are 
too  low  for  the  production  of  the  necessary  antibodies.  The 
same,  it  may  be  noted,  is  true  for  the  ordinary  tuberculin  test, 
if  it  were  ever  justifiable  to  use  it  in  such  cases. 

Of  the  value  of  the  test  there  is  no  doubt,  and  a  well-marked 
reaction  is  conclusive.  A  good  deal  of  difficulty  arises  from 
slight  and  doubtful  reactions,  and  it  sometimes  appears  to  fail  in 


382  TUBERCULOSIS 

cases  definitely  tuberculous.  A  positive  reaction  is  here  of  more 
value  than  a  negative  one. 

Calmette's  reaction  was  introduced  soon  after  Von  Pirquet's,  and 
consists  in  a  slight  conjunctivitis  and  oedema  of  the  caruncle,  with 
sero-fibrinous  discharge,  which  occurs  in  tuberculous  subjects  after 
the  instillation  of  a  drop  of  well-diluted  tuberculin  into  the  eye. 
Since  glycerin  has  an  irritant  action,  a  special  preparation  devoid 
of  this  substance  is  sold  for  the  test.  It  is  put  up  in  a  dry  form 
(being  precipitated  by  alcohol),  and  when  dissolved  in  water 
according  to  the  instructions  gives  a  dilution  corresponding  to 
i  :  100  or  i  :  200  of  the  original  tuberculin.  The  strength  has 
been  gradually  decreased  owing  to  calamitous  results  having  been 
obtained  when  the  stronger  dilutions  were  used.  It  should 
certainly  never  be  used  when  the  eyes  are  affected,  and  even 
when  they  are  healthy  I  personally  do  not  consider  the  risk  worth 
running  unless  the  diagnosis  is  of  great  importance,  and  all  other 
methods  have  been  tried  and  have  failed. 

2.  The  Opsonic  Index. — Numerous  observations  by  different 
observers  have  shown  that  in  health  the  opsonic  index  to  tubercle 
lies  between  0-8  and  1*2,  the  exceptions  being  very  rare  and  due 
perhaps  to  slips  in  the  technique.  As  a  matter  of  fact,  these  are 
outside  figures,  and  in  the  great  majority  of  cases  the  index  will 
be  found  to  lie  between  0*95  and  1*05.  If,  therefore,  the  diagnosis 
lies  between  health  and  tubercle,  an  index  below  0-8  or  above  1-2 
is  very  strong  evidence  in  favour  of  the  latter,  becoming  more 
convincing  the  remoter  it  is  from  these  figures,  either  above  or 
below.  Indices  between  0*8  and  1*2  are  not  of  much  value,  yet  a 
figure  of,  say,  0-85  on  several  occasions  is  very  suggestive. 

There  is  not  sufficient  evidence  as  yet  to  show  how  other 
diseases  influence  the  tuberculo-opsonic  index.  In  the  majority 
of  cases  the  figures  lie  within  the  normal  limits,  but  there  are 
occasional  exceptions.  In  general,  therefore,  the  results  given 
above  will  apply,  but  the  conclusions  are  less  certain. 

A  rapid  variation  of  the  index,  either  (a)  spontaneous,  or 
apparently  so,  or  (b)  due  to  auto-inoculation  from  exercise  or 
massage  of  the  lesion,  or  (c]  caused  by  an  injection  of  a  small 
dose  of  tuberculin,  is  much  more  suggestive — indeed,  practically 
diagnostic,  supposing  no  errors  are  made  in  the  determination. 
And  it  must  be  emphasized  that  when  the  opsonic  index  is 
required  for  diagnostic  purposes,  the  most  careful  technique  and 
attention  to  every  detail  is  absolutely  necessary. 


PRACTICAL   APPLICATIONS  383 

The  agglutination  reaction  has  been  recommended  as  a  means  of 
diagnosis,  but  it  is  not  always  present  at  any  stage  of  the  disease, 
and  there  are  practical  difficulties  in  the  way  of  its  determination. 
It  does  not  appear  to  have  come  into  general  use  even  in  France, 
where  much  attention  has  been  paid  to  it. 

Treatment. — The  essays  at  a  serum  treatment  have  been  many, 
and  no  method  has  attained  any  appreciable  degree  of  success. 
Good  results  have  been  obtained,  it  is  true,  by  several,  especially 
in  the  hands  of  their  inventors,  but  recent  work  by  Hort  and 
others  has  shown  that  normal  horse  serum  has  some  value  as  a 
curative  agent.  Maragliano's  serum  appears  to  be  prepared  by 
injecting  animals  with  various  extracts  of  tubercle  bacilli,  and 
is  supposed  to  be  both  bactericidal  and  antitoxic.  It  is  given 
either  alone  or  in  conjunction  with  a  vaccine.  McFarland 
prepared  an  antituberculin  by  immunizing  donkeys  for  long 
periods  with  tuberculin,  and  found  that  it  annulled  the  effects  of 
tuberculin  on  tuberculous  animals,  but  had  no  protective  or 
curative  powers  as  tested  on  guinea-pigs.  Clinical  evidence 
appeared  to  show  that  it  was  of  some  value. 

Marmorek's  toxin  is  quite  different  from  any  of  the  others, 
which  are  various  extracts  of  tubercle  bacilli,  or  of  their  soluble 
products.  It  is  prepared  by  cultivating  young  bacilli  (prepared 
in  such  a  way  that  they  form  homogeneous  emulsions)  in  a 
medium  containing  leucotoxic  serum,  prepared  by  injecting  calves 
with  guinea-pig  leucocytes.  It  is  supposed  to  be  the  actual 
toxin  which  is  developed  in  vivo.  This  substance  (which  is  of 
feeble  toxicity)  is  used  to  immunize  horses.  Some  good  results 
have  been  obtained,  but  perhaps  we  may  attribute  them  to  minute 
amounts  of  tuberculin  which  may  be  formed  in  the  cultures  and 
remain  unabsorbed  in  the  blood  of  the  horse  when  it  is  bled.  The 
experience  of  most  observers  has  not  been  favourable  to  its  use. 

The  other  sera  which  have  been  introduced  do  not  call  for 
further  notice. 

The  most  hopeful  method  of  combating  tubercle — other  than 
the  all-important  use  of  fresh  air,  good  food,  and  careful  regimen — 
consists  in  the  use  of  a  vaccine.  There  are  many  to  choose  from 
— old  tuberculin,  TR,  BE,  bovine  tuberculin,  oxytuberculin, 
antiphthisin,  etc.;  but  of  these,  Koch's  preparations  (old  tuber- 
culin, TR,  and  BE)  are  the  only  ones  in  general  use,  and  appear 
at  least  as  good  as  any. 

TR  (tuber culinum  residmtm)  is  prepared  by  triturating  living,  dry, 


384  TUBERCULOSIS 

virulent  tubercle  bacilli  by  a  mechanical  process  in  an  agate 
mortar.  The  powdered  bacilli  are  suspended  in  distilled  water 
and  centrifugalized.  The  supernatant  fluid  has  the  properties  of 
the  old  tuberculin,  and  is  called  TO  (O  =  obey,  =  upper).  The 
residue  is  dried,  and  again  powdered,  emulsified,  and  centri- 
fugalized, and  the  bacilli  are  now  found  to  have  been  reduced  to 
a  uniform  mass.  This  constitutes  TR,  which  thus  corresponds 
somewhat  to  an  endotoxin.  It  is  diluted  with  20  per  cent, 
glycerin,  and  when  diluted  it  is  advised  that  no  carbolic  acid 
should  be  added.  There  has  been  a  good  deal  of  confusion  with 
regard  to  the  dosage.  It  arose  from  the  fact  that  i  gramme  of  the 
dried  tubercle  bacilli  is  used  in  the  preparation  of  100  c.c.  of  the 
remedy,  so  that  each  i  c.c.  of  the  latter  contains  the  material 
derived  from  10  milligrammes  of  bacilli,  and  is  supposed  to  be 
equal  in  immunizing  power  thereto.  But  the  amount  of  dry 
residue  which  it  contains  is  only  one-fifth  of  this  amount,  the  fluid 
being  standardized  before  issue,  so  that  i  c.c.  contains  2  milli- 
grammes of  solid  substance.  The  dosage  adopted  in  this  book 
concerns  this  dry  residue  only. 

Tuberculins  are  also  prepared  from  bovine  tubercle  (perlsucht) 
bacilli,  and  its  use  mixed  with  the  human  form  (as  recommended 
by  Allen)  appears  rational  and  quite  worthy  of  a  trial.  The 
majority  of  cases  of  human  tubercle  are  due  to  bacilli  of  the 
human  type,  but  at  the  worst  the  material  derived  from  the  bovine 
bacilli  will  be  inert  and  do  no  harm. 

Another  substance  used  as  a  vaccine  is  Koch's  Bazillen- 
emulsion,  or  Neutuberculin,  usually  known  as  BE.  It  consists 
of  a  suspension  of  dried  and  ground  up  bacilli  in  equal  parts  of 
glycerin  and  water.  When  used  as  a  vaccine  it  is  diluted  with 
normal  saline  solution  and  heated  to  60°  C.  to  insure  sterility. 
This  may  also  be  done  with  TR.  It  contains  5  milligrammes 
solid  substance  per  c.c. 

As  regards  the  use  of  these  substances  :  We  may  recognize 
two,  or  perhaps  three,  methods — (i)  the  intensive  ;  (2)  the  opsonic, 
and  (3)  the  uncontrolled  use  of  small  doses. 

i.  In  the  intensive  method  the  idea  is  to  bring  about  as 
much  immunity  as  possible  as  quickly  as  possible,  by  the  use  of 
rapidly  increasing  doses,  taking  care  always  to  avoid  a  reaction. 
The  commencing  dose  is  very  small,  about  T\j-  milligramme 
(  =  TTrijoo  c-c-)  °f  °ld  tuberculin,  or  T-^^  milligramme  (of  solid 
substance)  of  TR.  The  dose  is  repeated  two  or  three  times  a 


PRACTICAL    APPLICATIONS  385 

week,  and  is  gradually  increased  until  500  milligrammes  (  =  J  c.c.) 
of  old  or  2  milligrammes  of  TR  is  given.  If  a  reaction  occurs 
an  interval  of  a  week  is  given,  and  the  treatment  recommenced 
with  a  smaller  dose.  The  dose  of  BE  may  be  the  same  as  that 
of  TR. 

There  are  numerous  slight  modifications  in  detail,  but  the  fore- 
going outline  will  serve  as  a  general  description  of  the  process  as 
it  is  used  in  many  of  the  Continental  clinics.  As  a  rule  no 
attempt  is  made  to  estimate  the  degree  of  immunity,  but  Koch 
suggests  the  agglutination  reaction  for  this  purpose.  It  usually 
rises  to  i  :  100,  or  thereabouts,  and  may  go  much  higher. 

As  a  result  of  the  treatment,  the  patient  is  certainly  immunized 
to  tuberculin,  since  he  fails  to  react  to  fairly  large  doses.  The 
process  is  then  stopped  for  a  time  and  recommenced.  The 
beneficial  effects  as  recorded  (as  I  have  seen  on  a  small  scale)  are 
undoubted,  and  the  risks  in  careful  hands  and  in  properly  selected 
cases  appear  to  be  slight. 

2.  The  opsonic  method  consists  in  the  use  of  such  doses  at  such 
intervals  as  will  cause  the  maximum  increase  in  the  opsonic 
index.  The  doses  here  are  very  much  smaller  than  in  the  last 
method.  TR  is  usually  employed,  and  the  amounts  range 
between  yo^Vc^  anc^  IWQV  milligramme.  The  idea,  of  course,  is 
to  avoid  the  possibility  of  a  summation  of  negative  phases,  and 
to  call  forth  the  greatest  possible  formation  of  protective  sub- 
stances. It  is  extremely  difficult  to  carry  out,  since  the  index 
alters  so  quickly  in  most  cases  of  progressive  tubercle  that  very 
frequent  determinations  of  the  index  are  necessary.  Perhaps  it 
is  the  ideal  method,  and  all  who  undertake  tuberculin  treatment 
on  a  large  scale  ought  to  acquire  some  experience  of  it,  but  con- 
siderations of  time  will  prevent  its  ever  coming  into  general  use 
as  a  routine  treatment.  In  practice  a  sort  of  modified  opsonic 
method  is  sometimes  used.  The  first  few  doses  are  controlled  by 
the  index,  and  the  optimum  dose  and  interval  determined.  These 
are  then  preserved  throughout  the  treatment,  with  perhaps 
occasional  determinations  of  the  index  at  times. 

It  must  be  pointed  out  that  the  theoretical  necessity  for  the 
opsonic  control  depends  on  the  acceptance  of  the  fact  that 
tuberculin  exerts  its  beneficial  influence  by  causing  an  increase 
in  the  amount  of  opsonins  present  in  the  blood.  But  this  is  not 
certain.  Admitting  that  the  immunity  is  due  to  antibodies,  these 
may  be  bactericidal  substances  or  anti-endotoxins,  and  we  have 


386  TUBERCULOSIS 

no  reason  whatever  for  thinking  that  the  opsonic  index  affords 
any  clue  to  the  amounts  of  these  substances  formed.  And,  of 
course,  as  is  held  by  many,  if  the  beneficial  effects  are  due  to 
repeated  slight  local  reactions,  acting  in  a  way  as  yet  not  under- 
stood, the  opsonic  index  is  of  little  value.  It  should  not  be 
forgotten  that  a  polynuclear  leucocyte  will  engulf  an  enormous 
number  of  tubercle  bacilli  in  a  short  time  even  when  the  opsonic 
index  is  low,  and  it  seems  improbable  that  it  ever  falls  so  low 
(even  when  the  negative  phase  is  most  marked)  as  to  prevent 
these  leucocytes  from  doing  their  work,  presuming  the  bacilli  are 
accessible.  A  high  opsonic  index  may  have  some  action  in 
preventing  generalization  of  the  bacilli— e.g.,  at  a  surgical  opera- 
tion— but  even  this  is  not  certain. 

It  must  be  noted  that  the  beneficial  effects  of  vaccine  treatment 
by  the  opsonic  control  are  not  denied,  but  that  the  opinion  is 
expressed  that  the  same  benefit^  may  be  obtained  by  simpler 
methods. 

Space  forbids  more  than  a  reference  to  the  use  of  auto- inocula- 
tion by  graduated  exercise,  massage,  etc.  This  is  supposed  to 
expel  some  bacilli  or  products  thereof  into  the  lymph  spaces,  etc., 
where  they  act  as  a  vaccine.  The  benefits  of  these  measures  is 
undeniable,  but  it  may  be  doubted  whether  it  is  due  entirely  or 
in  part  to  auto-inoculation,  and  it  would  appear  preferable  to 
employ  vaccines  in  known  doses. 

3.  The  use  of  small  doses  without  opsonic  or  other  control.  This 
combines  the  advantages  of  practicability  and  safety,  and  often 
gives  good  results.  How  they  compare  with  those  obtained 
by  the  other  methods  cannot  be  accurately  known.  Probably 
no  harm  has  ever  resulted  from  TT^Ty  milligramme  of  TR,  and  if 
this  amount  is  given  once  in  every  ten  days,  or  even  once  a  week, 
many  cases  improve  in  a  most  striking  manner.  My  experience 
has  been  mainly  confined  to  its  use  in  surgical  cases,  and  though 
I  have  met  with  numerous  disappointments  (which  I  believe  are 
not  unknown  with  the  most  strict  opsonic  control),  I  have  seen 
others  in  which  rapid  cure — e.g.,  of  a  chronic  sinus  or  tuberculous 
ulcer — has  been  obtained.  I  should  not  recommend  it  or  any 
other  method  of  vaccine  treatment  in  place  of  surgical  or 
sanatorium  treatment,  where  applicable ;  in  conjunction  with 
other  methods,  it  has  its  place. 

Preventive  Treatment. — This  is  at  present  of  but  little  importance 
in  human  pathology.  A  person  who  is  in  danger  of  becoming 


PRACTICAL   APPLICATIONS  387 

tuberculous  should  be  removed  from  the  infection  and  placed 
under  good  hygienic  conditions.  A  few  injections  of  TR  may  be 
given,  however,  and  analogy  with  a  similar  procedure  in  cattle 
would  lead  us  to  believe  that  it  may  be  of  some  value. 

Attempts  to  immunize  lower  animals  to  tubercle  have  been 
made  from  a  very  early  period  ;  it  was,  indeed,  the  discovery  of 
the  fact  that  dead  bacilli  are  absorbed  with  great  difficulty  that 
led  Koch  to  devise  his  TR.  The  vaccines  that  have  been  employed 
are  numerous — cultures  of  low  virulence,  dead  cultures,  avian 
and  reptilian  bacilli,  etc. — but  the  subject  entered  oh  a  new  and 
most  important  phase  in  1901,  when  von  Behring  published  his 
method,  which  is  of  great  theoretical  interest;  and  from  which 
important  practical  results  in  the  stamping  out  of  bovine  tubercle 
(so  great  a  pest  to  infants,  from  the  prevalence  of  bacilli  in  milk) 
have  been  esteemed  possible.  Here  the  vaccine  consists  of  living 
tubercle  bacilli  of  human  origin.  This  is  of  feeble  virulence  for 
cattle.  It  is  prepared  by  being  dried  in  vacuo,  emulsified  with 
glycerin  in  a  mortar,  and  diluted  with  normal  saline  solution 
containing  0*15  per  cent,  of  sodium  carbonate.  It  is  standardized 
in  such  a  way  that  i  c.c.  =  2  milligrammes  of  dried  bacilli.  The 
injections  are  made  intravenously;  two  are  given — the  first  of 
i  c.c.,  and  the  second,  about  twelve  weeks  later,  of  five  times  this 
amount. 

There  is  no  doubt  that  this  process  confers  a  certain  amount 
of  immunity,  or  rather  of  increased  resistance.  The  process  is 
harmless  to  calves,  but  sometimes  kills  older  animals  from 
pulmonary  oedema.  The  immunity  lasts  for  a  year  or  so. 

The  method  has  been  carefully  examined  at  Melun  by  Rossignol 
and  Vallee,  and  their  results  were  quite  favourable.  The 
vaccinated  animals  were  tested  (along  with  controls)  by  sub- 
cutaneous injection  of  bovine  tubercle,  by  injections  of  these 
bacilli  into  the  veins,  and  by  keeping  them  in  a  shed  with  animals 
known  to  be  tuberculous,  and  in  all  cases  a  greatly  increased 
degree  of  resistance  was  found.  But  Lignieres  pointed  out  a 
remarkable  fact,  that  animals  which  have  been  treated  in  this 
way  and  which  do  not  react  to  tuberculin,  and  are  apparently 
normal  at  the  autopsy,  nevertheless  may  contain  living  tubercle 
bacilli  in  their  lymphatic  glands.  This  is  of  extreme  interest  in 
connection  with  the  question  of  the  latency  of  bacilli  in  the  tissues. 

Von  Behring's  latest  vaccine  is  known  as  tuberculase,  or  tulase. 
It  appears  to  be  prepared  by  the  action  of  chloral  on  tubercle 

25—2 


388  TYPHOID    FEVER 

bacilli,  but  the  exact  details  are  not  available,  and  the  preparation 
is  not  yet  obtainable.  It  seems  not  to  contain  living  bacilli. 

Attempts  have  been  made  to  vaccinate  against  tubercle  (and  to 
treat  it  in  human  subjects)  by  ingestion.  Some  degree  of  success 
appears  to  have  been  obtained  by  feeding  calves  with  living 
human  tubercle  bacilli,  and  some  physicians  have  claimed  good 
results  by  administering  a  minute  dose  of  TR  by  the  mouth  ;  but 
in  what  way  these  methods,  so  uncertain  as  to  the  dose  which  is 
actually  absorbed,  are  preferable  to  the  use  of  the  svringe  is 
not  very  clear. 

This  is  a  very  brief  epitome  of  a  field  of  research  which  would 
require  many  volumes  for  its  adequate  treatment.  I  have 
attempted  to  give  the  main  essentials  only. 

Typhoid  Fever. 

The  pathogenic  action  of  the  typhoid  bacillus  appears  to  be  due 
entirely  to  the  action  of  an  endotoxin  which  is  set  free  when  the 
bacillus  undergoes  solution  in  the  body.  This  toxin  has  a  local 
and  a  general  action.  The  former  is  marked  in  the  regions  which 
harbour  the  bacillus,  and  in  which  it  undergoes  solution,  either 
by  autolysis  or  by  the  action  of  bacteriolysis—  the  Peyer's  patches, 
abdominal  lymph  glands,  spleen,  etc.  In  these  regions  it  produces 
hyperaemia,  followed  by  inflammatory  hyperplasia  of  the  lymphoid 
and  endothelial  cells,  which  diminish  the  blood-supply  and  may 
lead  to  necrobiosis  of  the  inflamed  tissues.  In  the  case  of  the 
Peyer's  patches  this  commonly  occurs,  the  lymphoid  tissue  being 
cast  off  as  a  slough,  and  the  wound,  becoming  infected  by  intestinal 
organisms,  may  extend  deep  into  the  bowel  wall  and  cause  haemor- 
rhage or  perforation.  The  process  is  less  likely  to  occur  in  the 
solid  organs,  which  are  nourished  by  blood  from  all  sides  and  in 
which  secondary  infections  are  less  frequent.  The  general  effects 
of  the  toxin — fever,  degenerations  of  the  kidney,  liver,  etc.— are 
not  characteristic.  The  failure  of  a  leucocytic  reaction  of  the 
bone-marrow,  and  consequent  leucopaenia,  is  worthy  of  notice. 

The  fact  that  typhoid  fever  is  a  septicaemia  and  that  the  living 
organisms  circulate  in  the  blood  must  not  be  forgotten. 

Immunity. — A  short  time  ago  typhoid  fever  was  regarded  as  a 
disease  in  which  the  immunity  was  purely  bacteriolytic.  During 
an  attack  of  the  disease  or  the  process  of  artificial  immunization  the 
amount  of  immune  body  (which  occurs  in  small  amounts  in 
health)  shows  a  steady  rise,  and  the  blood  soon  becomes  extremely 


PRACTICAL  APPLICATIONS  389 

potent  in  this  respect.  The  bacteriolytic  power  of  the  blood  as 
tested  by  Wright's  method  does  not  necessarily  show  a  great 
increase  ;  this  is  due  probably  to  the  lack  of  complement,  and  it  is 
possible  that  relapses  may  be  due  to  the  same  cause,  deviation  of 
complement  occurring  owing  to  the  excess  of  immune  body. 

There  is,  however,  no  doubt  that  phagocytosis  plays  a  part  of  the 
utmost  importance  in  the  natural  cure  of  the  disease,  and  that  the 
opsonic  content  of  the  blood  (as  tested  by  one  of  the  dilution 
methods)  undergoes  a  steady  rise  during  the  process  of  immuniza- 
tion. We  may  regard  the  mechanism  by  which  the  infection  is 
combated  as  a  double  one,  bacteriolysis  and  phagocytosis  being 
jointly  concerned.  That  the  latter  is  of  chief  importance  appears 
probable,  from  the  fact  that  typhoid  bacilli  set  free  their  endotoxin 
when  dissolved  by  means  of  bacteriolytic  sera. 

The  antitoxin  or  anti-endotoxin  of  typhoid  bacilli  can  be  readily 
prepared  from  animals  by  a  process  (somewhat  prolonged)  of 
immunization  with  the  endotoxin,  prepared  either  by  grinding  the 
bacilli,  by  dissolving  them  in  bacteriolytic  sera,  or  by  allowing 
them  to  undergo  aseptic  autolysis.  There  is,  however,  no  proof 
for  the  belief  that  this  substance  is  developed  during  the  natural 
process  of  cure,  or  that  it  is  a  cause  of  the  subsequent  immunity. 
It  is  quite  as  possible  that  the  cessation  of  the  febrile  process 
is  due  to  the  destruction  of  the  bacilli,  and  consequent  cessation 
of  the  supply  of  toxin. 

The  effect  of  the  true  typhoid  toxin  is  seen  during  the  earlier 
stages  of  the  disease,  when  the  temperature  is  continuous.  The 
intermittent  temperature  of  the  later  stages  is  probably  due  in 
part  to  the  absorption  of  other  toxins  from  the  ulcerated  surfaces 
of  the  Peyer's  patches,  and  in  part  to  the  liberation  of  endotoxins 
which  occurs  when  bacilli  are  dissolved  before  being  ingested. 

The  duration  of  the  immunity  after  a  natural  attack  of  the 
disease  is  not  accurately  determined,  but  it  is  certainly  long — 
perhaps  several  years.  That  due  to  preventive  inoculation  is 
thought  to  be  six  months  at  least,  but  here  again  exact  figures 
cannot  be  obtained.  The  duration  of  the  antibodies  produced  in 
typhoid  fever  varies.  The  agglutinins  usually  disappear  within 
one  or  two  years,  but  they  may  persist  for  seven  or  more.  The 
bacteriolysin  is  thought  to  go  sooner,  but  the  observations  on 
which  this  idea  is  based  were  mostly  on  the  bactericidal  powers 
of  the  blood,  and  open  to  fallacy. 

Diagnosis. — Usually  the  W7idal  reaction  is  all  that  is  required. 


39°  TYPHOID    FEVER 

The  standard  that  is  adopted  varies  in  different  laboratories,  but 
in  the  great  majority  of  cases  it  will  be  found  that  the  serum  at 
the  end  of  the  first  week  will  clump  in  a  dilution  of  i  :  30  in  one 
hour  at  the  body  temperature,  and  that  this  is  very  seldom  the 
case  in  health  or  in  other  diseases.  The  amount  of  agglutinin 
soon  increases,  and  may  reach  a  degree  at  which  it  will  agglu- 
tinate at  i  :  1,000  or  more.  The  naked-eye  reaction  as  carried  out 
by  a  modification  of  Wright's  method  renders  it  very  easy  to 
perform  exact  quantitative  work,  and  it  is  an  advantage  to  do  so  in 
all  cases  in  which  the  agglutination  does  not  reach  i  :  50  on  the 
first  examination,  since  a  rise  in  the  power  of  the  serum  is 
practically  diagnostic.  My  routine  method  is  as  follows  :  A  unit 
mark  is  made  about  i  inch  from  the  end  of  a  rather  wide 
Wright's  pipette,  and  with  this  9,  2,  4  and  9  units  of  a  watery 
emulsion  of  a  young  agar  culture  of  typhoid  bacilli  are  placed  in 
four  depressions  on  a  porcelain  slab  ;  dead  cultures  may  also  be 
used  in  the  same  way.  A  unit  of  serum  is  now  sucked  into  the 
pipette  and  mixed  with  the  first  pool  of  9  units  of  emulsion. 
One  unit  of  this  is  then  mixed  with  each  of  the  remaining  three 
pools,  the  process  being  carried  out  quickly,  so  as  to  avoid  the 
absorption  of  agglutinin  from  the  powerful  serum.  This  gives  a 
series  of  i  :  10,  i  :  30,  i  :  50,  and  i  :  100.  Each  pool  is  now  sucked 
into  a  Wright's  pipette  sealed  at  the  end,  and  incubated  at  37°  C. 
A  control  of  emulsion  without  serum  is  also  prepared  and  in- 
cubated. The  tubes  are  examined  at  the  end  of  one  hour,  and 
agglutination,  if  present,  is  most  obvious. 

The  diagnosis  might  also  be  made  from  a  determination  of  the 
opsonic  index  by  the  dilution  method,  or  of  the  amount  of  immune 
body,  but  these  processes  are  much  more  tedious. 

Where  a  very  early  diagnosis  is  required  (i.e.,  before  the 
appearance  of  the  agglutination  reaction)  the  bacilli  may  be 
sought  for  in  the  stools  or  blood.  This  was  formerly  difficult,  but 
it  has  been  greatly  facilitated  by  the  introduction  of  new  methods 
involving  the  use  of  special  culture  media,  in  which  the  bacilli 
grow  rapidly  and  form  characteristic  colonies.  These  are  beyond 
the  scope  of  this  work,  and  a  description  of  them  will  be  found  in 
Hewlett's  "  Bacteriology,"  second  edition. 

Treatment.— The  curative  treatment  is  not  satisfactory.  The 
use  of  a  simple  bacteriolytic  serum  is  useless  or  even  dangerous, 
for  the  reasons  given  earlier.  The  best  hope  for  the  future  is 
in  the  use  of  an  anti-endotoxic  serum,  such  as  was  prepared  by 


PRACTICAL    APPLICATIONS  3QI 

Allan  Macfadyen  and  others,  and  is  now  put  on  the  market  by 
Messrs.  Burroughs  and  Wellcome,  under  the  supervision  of 
Professor  Hewlett.  Some  promising  results  have  already  been 
obtained  by  its  use,  and  it  seems  to  be  quite  harmless.  It  seems 
only  to  be  of  advantage  when  used  in  the  early  stages  of  the 
disease,  as  is  naturally  the  case  with  any  serum  which  acts  by 
neutralizing  a  soluble  toxin. 

Chantemesse  has  apparently  attained  a  very  high  degree  of 
success  by  the  use  of  a  serum  obtained  by  injecting  horses  with  a 
soluble  toxin,  prepared  by  cultivating  the  bacillus  in  an  extract  of 
spleen  digested  with  pepsin  and  subsequently  neutralized.  It  is 
very  doubtful  that  this  is  the  genuine  toxin,  since  it  is  feeble  in 
action  and  not  destroyed  at  100°  C.  The  serum  thus  obtained  is 
used  in  very  small  doses,  and  appears  to  act  (as  pointed  out  by 
Wright)  rather  as  a  vaccine  than  as  a  means  of  conferring  passive 
immunity.  The  results,  however,  appear  to  have  been  excellent, 
but  the  serum  is  not  yet  obtainable  commercially. 

Typhoid  fever  has  also  been  treated  by  the  use  of  small  doses 
of  vaccine,  and  some  promising  results  obtained  ;  but  the  method 
is  still  on  its  trial. 

Preventive  Treatment. — The  earliest  method  was  that  of  Wright, 
and  it  has  probably  not  been  surpassed.  It  consists  in  the  use  of 
vaccines  of  typhoid  bacilli  cultivated  in  broth  for  twenty-four 
hours,  killed  at  60°  C.,  counted  by  admixture  with  red  corpuscles, 
and  preserved  by  means  of  lysol  or  carbolic  acid.  Two  doses  are 
given,  the  first  being  750  to  1,000  millions,  the  second  twice  this 
amount,  the  injection  being  usually  made  deep  in  the  subcutaneous 
tissue  of  the  flank.  The  injections  are  given  at  intervals  of  one 
or  two  weeks.  Local  and  general  symptoms  of  some  severity 
follow  :  redness  and  swelling  at  the  site  of  inoculation,  lymphan- 
gitis, etc.,  with  fever,  headache,  and  general  malaise.  These  last 
but  a  short  time,  and  no  evil  consequences  have  been  recorded. 
It  is  necessary,  however,  for  the  patient  to  be  prepared  to  stay 
in  bed  during  the  day  of  inoculation  and  the  next  day  :  the 
unpleasant  effects  may  be  reduced  to  a  minimum  by  the  simul- 
taneous administration  of  chloride  of  calcium  by  the  month 
(15  to  45  grains). 

As  a  result  of  these  injections,  protective  substances,  and  espe- 
cially agglutinins,  make  their  appearance  in  the  blood,  and  may 
persist  for  several  years.  These  may  be  taken  as  affording  some 
test  as  to  the  degree  of  immunity  conferred,  and  as  to  its  duration. 


392  TYPHOID   FEVER 

Space  does  not  permit  us  to  discuss  at  length  the  evidence  in 
favour  of  this  method  of  inoculation.  It  has  been  strongly,  and 
perhaps  unfairly,  opposed  in  certain  quarters,  but  a  careful  and 
unprejudiced  study  of  the  statistics  of  the  British  Army  in  South 
Africa  and  elsewhere  seems  to  render  it  quite  clear  that  the  process 
develops  a  real  though  not  absolute  protection  against  an  attack 
of  the  disease,  and  is  still  more  valuable  in  diminishing  the 
mortality  rate  amongst  those  attacked.  The  analysis  of  these 
figures  is  by  no  means  easy,  but  certain  isolated  cases  are  in 
themselves  very  convincing.  As  an  example  (one  out  of  many) 
we  may  quote  the  Manchester  Regiment,  in  which,  out  of 
200  men  inoculated  there  were  3  cases,  without  a  death,  whilst  of 
the  517  uninoculated  23  were  attacked  and  3  died.  In  general 
terms  we  may  say  that  the  mortality  from  an  attack  fell  from 
over  12  per  cent,  in  the  uninoculated  to  below  6  per  cent,  in  the 
inoculated,  taking  the  results  from  the  whole  of  the  Transvaal 
and  Natal.  One  caution  is  necessary :  the  first  result  of  the  in- 
jection is  the  production  of  a  negative  phase  okjncreased  suscep- 
tibility, and  injections  should  not  be  practised,  if  avoidable,  when 
the  patient  is  already  exposed  to  the  infection. 

The  vaccine  is  prepared  by  the  Lister  Institute,  and  can  be 
bought  ready  for  use. 

Numerous  modifications  of  the  process  have  been  adopted. 
Pfeiffer  and  Kolle  prepare  their  vaccine  from  agar  cultures. 
Bassenge  and  Rimpau  do  the  same,  but  use  very  small  doses — 
TJ  to  f  milligramme.  Friedberger  and  Moreschi  give  intra- 
venous injections  of  infinitesimally  small  doses  of  bacilli  killed  at 
120°  C.  Wassermann's  vaccine  is  prepared  by  killing  a  culture  at 
60°  C.,  allowing  it  to  undergo  autolysis  at  37°  C.  for  five  days, 
filtering,  and  desiccating  in  vacua  at  35°  C.  It  forms  a  yellowish - 
white  powder,  of  which  the  dose  is  0*0017  gramme,  equal  to 
12  milligrammes  of  the  culture.  Numerous  other  methods  might 
be  enumerated,  but  they  have  mostly  been  investigated  on  a  small 
scale  only,  and  Wright's  vaccine  is  of  proved  efficacy  and  easy  to 
prepare. 

Antityphoid  serum  has  often  been  employed,  and  in  all  proba- 
bility is  of  value  in  that  it  affords  a  rapid  (or  practically  instan- 
taneous) means  by  which  immunity  can  be  produced,  and  eliminates 
the  negative  phase  altogether.  The  serum  is  prepared  from  the 
horse,  which  is  injected  with  gradually  increasing  doses  of  dead 
bacilli,  and  subsequently  of  living  ones.  It  can  be  obtained  of 


PRACTICAL   APPLICATIONS  393 

great  potency,  as  determined  by  its  power  of  agglutination,  which 
may  be  manifested  when  it  is  diluted  a  million  times.  But  the 
immunity  it  confers  (judging  from  laboratory  experiments  and 
analogy  with  other  diseases)  is  but  temporary,  and  the  method 
can  only  be  of  occasional  value. 

The  plan  of  injecting  the  serum  and  vaccine  in  combination 
suggested  by  Besredka  is  more  promising.  It  is  carried  out  as 
follows  :  A  twenty-four-hour  agar  culture  of  bacilli  is  mixed  with 
typhoid  serum  and  incubated  at  37°  C.  for  twenty-four  hours. 
The  bacilli  are,  of  course,  agglutinated,  and  the  mass  is  washed 
free  from  serum  by  repeated  centrifugalizations  with  sterile 
normal  saline  solution.  The  bacilli  are  then  suspended  in  this 
solution  and  heated  to  60°  C.  for  one  hour.  This  vaccine  is  said 
to  produce  very  rapid  immunity,  with  practically  no  negative 
phase,  and  the  local  and  general  reactions  it  produces  are  but 
slight.  It  has  not  yet  been  tested  on  a  large  scale. 

Simultaneous  injections  of  serum  and  vaccine  have  also  been 
employed  by  Calrpette  and  others.  The  results  do  not  appear  to 
be  as  satisfactory  as  those  obtained  from  the  use  of  the  vaccine 
alone. 

Bacillus  Coli. 

The  Bacillus  coli,  in  addition  to  its  haemolysin  (colilysin)  already 
mentioned,  produces  an  endotoxin,  on  which  its  pathogenic  action 
doubtless  depends.  Its  vaccine  is  moderately  toxic,  causing 
severe  local  irritation  and  general  febrile  reaction  if  the  initial 
dose  be  a  large  one.  Filtrates  from  broth  cultures  are  practically 
devoid  of  toxicity. 

In  regard  to  its  immunity  reactions  B.  coli  closely  resembles 
B.  typhosus.  Bacteriolytic  and  opsonic  substances  are  developed 
during  the  course  of  the  disease,  and  in  both  cases  the  opsonic 
index  must  be  estimated  by  the  dilution  method,  Wright's  original 
process  giving  inaccurate  results.  There  is  some  difference  with 
regard  to  the  production  of  the  agglutination  reaction,  which  is 
invariably  present  in  typhoid  infections,  except  in  those  acute 
cases  of  the  disease  in  which  the  patient  dies  before  it  has  time  to 
develop.  In  infections  with  B.  coli  this  is  not  the  case,  and  in 
many  of  the  local  infections,  such  as  cystitis,  pyelitis,  etc,,  no 
appreciable  agglutinative  properties  are  developed.  When  animals 
are  immunized  with  massive  doses  extremely  powerful  clumping 
sera  are  obtained,  but  this  is  not  always  observed  in  the  treatment 
of  the  human  patient  with  vaccines. 


394  BACILLUS    COLI 

The  main  difference,  pathologically,  between  the  two  organisms 
is  that,  whereas  the  disease  due  to  the  typhoid  bacillus  is  usually 
a  septicaemia,  localized  infections  (typhoid  osteitis,  etc.)  only 
occur  as  sequelae,  the  diseases  due  to  B.  coli  are  almost  in- 
variably local  inflammations,  blood  infections,  except,  perhaps,  as 
mere  terminal  phenomena,  being  rare  in  the  extreme.  These 
inflammatory  lesions  are  extremely  common  and  important,  and 
of  most  diverse  nature.  The  majority  are  in  connection  with 
the  urinary  (cystitis,  pyelitis,  etc.)  and  alimentary  systems 
(cholangitis,  cholecystitis,  appendicitis,  etc.).  It  also  causes 
acute  peritonitis,  but  usually  in  association  with  other  bacteria. 
It  may  affect  almost  any  part  of  the  body,  causing  broncho- 
pneumonia,  otitis  media,  endometritis,  metritis,  and  a  host  of 
other  diseases. 

The  only  specific  treatment  of  any  avail  is  the  use  of  a  vaccine. 
A  serum  has  been  prepared,  but  appears  to  be  quite  useless. 
The  vaccine  should  always  be  prepared,  if  possible,  from  the 
patient's  own  culture,  since  various  strains  classified  as  B.  coli 
differ  slightly  the  one  from  the  other.  In  addition  to  this,  there 
are  very  marked  differences  in  virulence,  cultures  isolated  from 
the  stools  being  in  general  less  virulent  than  those  derived  from 
the  diseased  tissues.  In  cases  in  which  treatment  has  to  be  pro- 
longed it  is  sometimes  an  advantage  to  prepare  fresh  vaccines 
from  time  to  time,  since  the  organism  may  change  its  type  to 
accommodate  itself  to  the  immune  substances  produced. 

The  initial  doses  should  be  small  (10  to  40  millions),  and  it  will 
be  rarely  found  advisable  to  exceed  250  millions.  Too  large  an 
initial  dose  may  be  badly  tolerated,  causing  rise  of  temperature 
and  a  good  deal  of  local  reaction,  but  no  permanent  bad  results 
seem  to  have  been  observed.  On  the  other  hand,  it  may  some- 
times be  noted  that  in  severe  febrile  cases  the  vaccine  acts  more 
like  an  antitoxin,  causing  a  sudden  drop  in  the  temperature  and 
a  rapid  amelioration  of  the  symptoms,  which  may  or  may  not  be 
associated  with  a  great  improvement  in  the  local  lesion.  This  is 
shown  in  the  accompanying  chart,  from  a  severe  case  of  cystitis 
under  the  care  of  Mr.  Burghard  at  King's  College  Hospital.  As 
far  as  his  general  condition  was  concerned,  he  was  practically  well 
within  ten  days  of  the  commencement  of  the  treatment.  The 
amount  of  pus  in  the  urine  fell  to  less  than  an  eighth  of  its 
original  volume,  but  had  not  disappeared  entirely  when  he  was 
discharged.  This  has  been  my  usual  experience  of  cystitis  due 


PRACTICAL   APPLICATIONS 


395 


to  B.  coli — vaccine  treatment  cures  all  the  symptoms  and  reduces 
the  pus  and  bacilli  present  in  the  urine  to  a  small  fraction  of  its 
original  amount,  but  fails  to  remove  them  entirely.  Other 
observers  have  been  more  fortunate  (Wright,  Western,  Norton, 
and  others),  but  I  believe  my  experience  is  the  general  one,  and 
that  complete  cures  are  unusual.  There  is  no  doubt,  however, 
that  the  treatment  is  the  best  available,  the  disease  being 
notoriously  resistant  to  simple  methods  of  treatment.  If  the 
injections  are  made  with  opsonic  control,  the  dilution  method  of 


FIG.  71. — CHART  FROM  A  SEVERE  CASE  OF  CYSTITIS  DUE  TO  B.  COLI. 

The  continuous  line  shows  the  temperature,  the  dotted  line  the  amount  of 

pus  in  the  urine. 

estimating  the  index  should  be  used.  In  default  of  this,  the 
injections  may  be  given  every  eight  to  twelve  days,  doubling  the 
dose  until  the  upper  limit  is  reached. 

The  treatment  of  inflammatory  lesions  of  other  regions  (chole- 
cystitis, persistent  sinuses,  etc.)  appears  to  be  more  satisfactory, 
remarkable  cures  having  been  obtained.  Butler  Harris  has 
obtained  good  results  in  endometritis,  cervical  catarrh,  and 
mucous  colitis.  In  the  former  disease  he  gives  a  small  dose  a 
week  before  and  after  each  menstrual  period. 

It  is  in  infections  due  to  B.  coli  that  some  of  the  most  striking 


3Q6  DYSENTERY 

successes  of  vaccine  therapy  have  been  achieved.  It  has  been 
used  in  some  extremely  severe  conditions  with  profound  toxaemia, 
and  in  no  case  did  it  render  the  patient's  condition  worse.  Small 
doses  should,  of  course,  be  used  in  such  cases,  but  the  severity  of 
the  disease  need  be  no  bar  to  the  use  of  the  remedy. 

Dysentery. 

Bacillary  dysentery,  under  which  heading  we  may  include  some 
at  least  of  the  cases  known  as  ulcerative  colitis,  asylum  dysentery 
and  infantile  diarrhoea,  is  in  its  general  pathology  closely  akin  to 
typhoid  fever.  Its  toxin  is  an  endotoxin  which  is  only  set  free 
when  the  bacilli  undergo  solution,  and  its  cure  is  associated  with, 
and  perhaps  due  to,  the  development  of  a  large  amount  of  bacterio- 
lysin.  The  main  difference  between  the  two  diseases  is  that, 
whereas  typhoid  fever  is  a  self-limited  disease,  in  which  the  patient 
either  dies  or  immunizes  himself  in  a  fairly  constant  period  after 
infection,  in  dysentery  the  disease  has  a  tendency  to  become 
chronic,  the  degree  of  immunity  produced  being  sufficient  to  slow 
the  course  of  the  disease,  but  insufficient  to  arrest  it  altogether. 
The  bacilli  are  in  the  main  limited  to  the  intestinal  lesions,  but 
the  fact  that  they  have  been  found  in  the  heart-blood  of  a  new- 
born foetus  whose  mother  was  suffering  from  the  disease  leads  us 
to  believe  that  they  may  circulate  in  the  blood  ;  in  most  cases 
they  are  probably  quickly  destroyed,  for  normal  serum  has  some 
bacteriolytic  action. 

The  toxin  is  contained  in  the  bodies  of  the  organisms,  which 
are  much  more  poisonous  than  those  of  typhoid  bacilli.  It  may 
be  obtained  by  Macfadyen's  method  of  grinding  at  the  temperature 
of  liquid  air,  by  aseptic  autolysis  at  37°  C.,  or  simply  by  filtering 
old  broth  cultures  in  which  some  of  the  bacilli  have  undergone 
solution.  Conradi's  toxin,  prepared  by  submitting  emulsions  to 
autolysis  for  forty-eight  hours,  was  fatal  to  rabbits  in  doses  of 
i  c.c.,  causing  diarrhoea,  subnormal  temperature,  etc.  According 
to  Todd,  the  toxin  is  developed  best  in  alkaline  broth,  and  attains 
its  maximum  in  about  six  weeks,  after  which  time  it  begins  to 
diminish  in  potency.  It  is  more  thermostable  than  the  exotoxins, 
resisting  heating  to  70°  C.  for  one  hour,  and  only  being  destroyed 
slowly  at  the  boiling-point.  It  is  possible  to  obtain  an  anti-endo- 
toxin,  but  the  treatment  of  the  animals  has  to  be  conducted  with 
great  care.  It  is  probable  also  that  the  serum  of  animals 
immunized  to  the  bacilli  themselves  contains  some  antitoxic 


PRACTICAL   APPLICATIONS  397 

properties,  especially  if  the  bacilli  are  injected  into  the  veins 
(Besredka).  In  preparing  this  serum  it  is  a  great  advantage  to 
follow  Besredka's  process,  and  make  use  of  sensitized  bacilli.  If 
the  unaltered  bacilli  are  used  the  process  presents  some  difficulties, 
especially  in  the  case  of  small  animals. 

The  serum  thus  prepared  possesses  powerful  agglutinating  and 
bacteriolytic  properties,  and  is  extremely  powerful  as  a  prophylactic 
agent.  Kruse's  serum  protected  a  guinea-pig  against  a  lethal 
dose  of  the  bacilli  in  doses  of  ginn^  gramme>  and  ToiiRr  c-c-  °f 
Shiga's  serum  (activated  with  a  suitable  amount  of  complement) 
would  sterilize  i  c.c.  of  a  twenty-four-hour  broth  culture  of  the 
bacilli. 

Antidysentery  serum  has  now  fully  proved  its  value  in  the 
treatment  of  acute  dysentery.  According  to  Shiga,  it  reduces  the 
mortality  of  the  disease  by  nearly  50  per  cent.  Kruse  claims  that 
his  serum  causes  a  rapid  diminution  in  the  number  of  the  stools, 
such  as  is  effected  by  no  other  agent,  a  general  improvement  in 
the  patient's  condition,  a  shortening  of  the  convalescence,  and  a 
diminution  of  the  mortality.  Large  doses,  frequently  repeated, 
are  required. 

It  has  probably  a  complex  action,  being  at  the  same  time 
antitoxic  and  bacteriolytic,  and  contains  opsonins  and  bacterio- 
precipitins. 

In  chronic  cases  it  is  of  much  less  value,  and  in  these  forms 
reliance  must  be  placed  on  vaccine  therapy.  This  has  been  care- 
fully studied  by  Captain  Forster  and  others.  In  Forster's  patients 
the  mortality  fell  from  6-3  per  cent,  to  0-9  per  cent.,  several  cases 
of  the  extremely  chronic  type  which  defies  all  ordinary  treatment 
for  years  being  completely  cured.  He  uses  no  opsonic  control, 
and  standardizes  his  vaccines  by  determining  the  minimal  lethal 
dose  ;  this  is  necessary,  since  the  various  strains  differ  greatly  in 
toxicity.  If  the  minimal  lethal  dose  for  a  rabbit  is  about  0*4  c.c., 
doses  of  o'i,  0-2,  0-3,  and  0-4  c.c.  are  given  at  intervals  of  about  ten 
days.  If  symptoms  of  an  overdose  are  produced,  the  amount  given 
at  the  next  injection  is  reduced. 

Prophylactic  treatment  by  injections  of  killed  cultures,  either 
as  they  are  or  after  autolysis  or  sensitization  with  an  immune 
serum,  or  injected  in  conjunction  with  immune  serum,  have  been 
used  on  a  large  scale  by  Shiga  and  others,  with  apparent  good 
results  as  far  as  the  case  mortality  is  concerned,  though  with  less 
obvious  effect  on  the  prevalence  of  the  disease.  This  phenomenon 


3Q8  CHOLERA 

is  readily  intelligible  if  we  regard  the  subsequent  immunity  as 
being  a  condition  in  which  the  body,  having  been  once  trained  to 
do  so,  readily  manufactures  antibodies  and  other  protective  sub- 
stances when  infection  occurs.  Since  no  protective  substances 
are  present  at  the  time,  infection  occurs  as  in  a  normal  person, 
but  the  defensive  substances  are  very  quickly  produced.  Shiga's 
vaccine  was  prepared  by  emulsifying  a  twenty-four-hour  agar 
culture  of  the  bacillus  in  5  c.c.  of  normal  saline,  heating  to  60°  C. 
for  one  hour,  and  submitting  the  dead  bacilli  to  autolysis  at  37°  C. 
for  two  days.  It  is  then  filtered  and  used  in  doses  of  0^05  to  0-5  c.c. 
The  serum  becomes  strongly  agglutinating  and  bactericidal. 
Other  methods  have  been  proposed. 

Immunity  reactions,  especially  that  of  agglutination,  are  of 
great  value  in  the  diagnosis  of  dysentery,  and  especially  of  the 
type  of  the  bacillus  present.  In  acute  cases  this  is  hardly 
necessary,  since  modern  methods  have  rendered  the  task  of 
isolating  the  bacilli  from  the  stools  an  easy  one.  In  the  chronic 
forms  this  is  extremely  difficult,  and  recourse  must  be  had  to  the 
agglutination  test.  The  reaction  is  not  as  strong  as  in  typhoid 
fever,  a  positive  result  at  a  dilution  of  i  :  50  being  diagnostic. 
The  blood  should  be  tested  against  any  strains  of  dysentery 
bacilli  which  may  be  available,  especially  if  vaccine  treatment  is 
to  be  used. 

The  method  of  absorption  of  complement  has  also  been  used. 

Cholera. 

In  cholera  the  living  organisms  are  strictly  limited  to  the 
intestinal  contents,  and  the  disease  appears  to  be  a  pure  intoxica- 
tion, without  access  of  living  bacteria  to  the  tissues.  It  is,  how- 
ever, probable  that  this  is  not  the  case,  and  that  the  vibrios  enter 
the  blood  and  there  suffer  rapid  and  complete  bacteriolysis,  their 
endotoxins  being  liberated  in  the  process.  But  there  is  nothing 
that  can  be  called  a  local  lesion,  and  the  disease  is  not  a  septi- 
caemia in  the  ordinary  sense  of  the  word. 

The  toxin  of  cholera  is  a  typical  endotoxin.  The  nitrates  from 
broth  cultures  are  of  very  feeble  toxicity,  though  they  possess 
immunizing  properties,  due  doubtless  to  some  degree  of  autolysis 
which  has  taken  place,  and  to  the  presence  of  free  receptors. 
Bacilli  killed  with  agents  such  as  chloroform  or  thymol  are  highly 
toxic,  especially  if  injected  along  with  an  immune  serum,  so  that 
they  can  be  rapidly  dissolved.  The  endotoxin  can  be  prepared 


PRACTICAL   APPLICATIONS  399 

by  aseptic  autolysis,  or  by  the  freezing  and  grinding  method  of 
Macfadyen.  In  either  case  it  is  thermolabile,  being  largely 
destroyed  at  60°  C.,  so  that  cultures  from  which  the  toxin  is  to  be 
prepared  must  not  be  killed  by  heat.  Metchnikoff  and  others 
claim  to  have  produced  a  soluble  exotoxin  by  the  use  of  very 
virulent  cultures  in  broth  :  it  is  thermostable  and  not  very 
potent.  An  antitoxin  to  it  was  prepared,  but  only  very  low  grades 
of  potency  were  obtainable.  Macfadyen's  toxin  was  much  more 
toxic,  and  an  anti-endotoxin  of  high  potency  was  obtainable. 
There  is  no  demonstrable  antitoxin  in  the  ordinary  bacteriolytic 
serum  obtained  by  the  immunization  of  animals  to  the  bodies  of 
the  bacilli,  or  in  the  serum  of  cholera  convalescents. 

Cholera  presents  the  best  example  of  an  apparently  pure 
bacteriolytic  immunity,  and  presents  a  good  example  of  the 
difficulties  inherent  in  the  explanation  of  this  subject.  The 
serum  of  an  immunized  animal  or  that  of  a  person  who  has 
recently  recovered  from  cholera  is  powerfully  bacteriolytic,  giving 
Pfeiffer's  phenomenon  in  its  earliest  discovered  and  most  marked 
form :  it  is  almost  the  only  organism  which  is  completely  dissolved 
in  vitro  under  suitable  conditions.  Such  a  serum  is  also  strongly 
protective,  shielding  animals  against  several  times  the  lethal  dose 
of  living  vibrios,  and  it  seems  difficult  to  avoid  the  conclusion 
that  its  preventive  properties  are  due  to  its  bacteriolytic  action. 
But  this  is  very  difficult  to  maintain  in  view  of  the  fact  that  the 
serum  increases  the  toxic  effect  of  dead  vibrios  (and  under  some 
circumstances  of  living  ones),  owing  to  the  liberation  of  endotoxin. 
It  seems  rather  as  if  the  presence  of  bacteriolytic  substances  is 
actually  harmful  to  the  animal,  allowing  the  organisms  to  set  free 
their  toxin,  instead  of  being  taken  up  by  the  phagocytes  and 
remaining  harmless.  I  am  not  aware  that  any  opsonic  experi- 
ments by  the  dilution  method  (which  alone  would  be  of  value) 
have  been  carried  out. 

Diagnosis. — In  dealing  with  cases  of  supposed  sporadic  cholera 
the  main  problem  is  the  recognition  of  the  vibrio  isolated  from 
the  stools,  usually  an  easy  matter.  The  morphological  and 
cultural  characters  will  of  course  afford  great  help,  but  they  take 
some  time  to  work  out,  and  more  reliance  is  to  be  placed  on  the 
immunity  tests,  which  are  quicker  and  more  conclusive.  The 
agglutination  reaction  is  most  convenient,  and  can  be  carried  out 
on  the  dejecta  themselves,  if  the  suspected  organisms  are  present 
in  large  numbers.  Some  of  the  mucus  is  broken  up  in  a  little 


400  CHOLERA 

peptone  solution,  and  two  hanging-drop  preparations  are  made, 
one  with  the  addition  of  normal  serum  in  i  :  50  dilution,  the 
other  with  a  i  :  500  dilution  of  a  powerful  anticholera  serum, 
such  as  can  be  obtained  commercially.  Cholera  vibrios  become 
paralyzed  and  agglutinated  in  the  second  specimen,  not  in  the 
first.  When  smaller  numbers  are  present  a  culture  (probably 
impure,  but  with  the  vibrios  in  sufficient  abundance  to  serve  for 
the  test)  may  be  made  by  incubating  peptone-water  inoculated 
with  a  flake  of  mucus  for  eight  to  twelve  hours.  This  is  to  be 
tested  with  the  serum  in  the  ordinary  way,  and  should  agglutinate 
at  nearly  the  same  dilution  as  a  known  cholera  culture.  The 
serum  should  be  a  powerful  one,  clumping  at  i  :  10,000  or 
more. 

The  Pfeiffer's  reaction  is  perhaps  more  conclusive,  and  is 
carried  out  as  follows  :  The  test  immune-serum  is  diluted  with 
broth  or  normal  saline,  so  that  i  c.c.  contains  o-ooi  c.c.  of  serum; 
i  c.c.  of  this  fluid  is  used  to  emulsify  a  loopful  of  a  young  agar 
culture  of  the  suspected  organism,  and  the  emulsion  injected 
intraperitoneally  into  a  young  guinea-pig.  After  a  few  minutes  a 
little  peritoneal  fluid  is  withdrawn  by  means  of  a  capillary  tube, 
and  the  vibrios  will  be  seen  to  have  become  non-motile,  and  to  be 
undergoing  the  characteristic  change  into  slightly  refractile 
rounded  masses.  After  a  short  time  more  they  will  be  found 
to  have  disappeared  altogether.  A  control  experiment  with 
normal  serum  may  be  made.  This  test  is  of  great  value,  many 
closely  allied  organisms  failing  to  react.  But  no  test  is  absolutely 
conclusive,  since  a  few  cultures  (notably  the  El  Tor  vibrio)  have 
been  found  to  give  all  or  most  of  them,  and  yet  have  been  isolated 
in  a  region  in  which  cholera  is  not  known  to  occur.  The  subject 
is  not  yet  settled,  but  in  the  meantime  the  probability  that  any 
organism  which  reacts  positively  to  the  agglutination  and 
Pfeiffer's  tests  is  true  cholera  is  enormous. 

The  serum  of  persons  convalescent  from  cholera  agglutinates 
the  vibrios  at  dilutions  of  i  :  100  or  more  for  some  months  after 
the  attack,  a  fact  which  may  be  of  some  value  in  determining 
the  nature  of  a  previous  disease  and  a  possible  immunity  to 
cholera. 

As  regards  treatment,  the  ordinary  bacteriolytic  serum  is  quite 
useless,  and,  as  far  as  I  am  aware,  no  potent  anti-endotoxic  serum 
has  been  tried.  The  prophylactic  treatment  is  on  a  sounder 
footing.  It  was  introduced  by  Ferran,  of  Barcelona,  as  early  as 


PRACTICAL   APPLICATIONS  40! 

1884,  very  soon  after  the  discovery  of  the  V.  cholera  by  Koch. 
His  results  were  of  doubtful  value,  his  vaccines  being  made  of 
cultures  of  feeble  virulence,  and  perhaps  impure.  The  method 
was  placed  on  a  scientific  basis  by  Haffkine,  who  showed  the 
necessity  for  the  use  of  cultures  of  great  virulence.  These  are 
prepared  by  passage  through  guinea-pigs.  A  more  than  lethal 
dose  of  a  laboratory  culture  is  injected  into  the  peritoneum  of  a 
guinea-pig,  and  the  peritoneal  fluid  (rich  in  vibrios)  is  collected 
after  death.  This  fluid  is  incubated  in  a  thin  layer,  so  as  to  allow 
of  thorough  aeration,  for  fifteen  hours,  and  is  then  administered 
intraperitoneally  into  a  second  animal.  After  about  twenty  or 
thirty  passages  the  culture  will  have  attained  its  maximum 
virulence,  the  lethal  dose  being  some  ^  of  the  original.  Its 
potency  falls  off  in  some  ten  days,  and  a  few  further  passages  are 
required  to  restore  it. 

The  treatment  is  commenced  by  a  dose  of  attenuated  virus. 
This  is  prepared  by  cultivating  an  ordinary  laboratory  stock  in 
broth  at  39°  C.  in  conditions  of  complete  aeration.  An  inocula- 
tion on  agar  is  made  every  day,  until  (after  a  few  days)  the  fluid 
is  found  to  be  sterile.  The  process  is  now  recommenced,  using 
the  last  agar  culture  that  grew,  and  after  several  generations  a 
culture  of  very  feeble  virulence  is  obtained.  It  causes  oedema, 
but  no  necrosis,  when  injected  under  the  skin.  The  vaccines  are 
prepared  by  cultivating  the  organisms  on  agar  slants  of  definite 
size  (10  centimetres  long)  for  twenty-four  hours,  and  emulsify- 
ing with  8  c.c.  of  broth,  or  6  c.c.  of  0-5  per  cent,  solution  of 
carbolic  acid.  The  dose  is  i  c.c.  One  or  two  injections  of  the 
attenuated  virus,  followed  by  one  of  the  exalted,  all  at  intervals 
of  three  to  five  days,  may  be  given,  or  the  exalted  virus  only  may 
be  used.  The  injections  cause  moderate  fever,  headache,  and 
general  malaise,  and  local  tenderness,  swelling  and  enlargement  of 
the  corresponding  lymph  glands,  all  of  which  pass  off  in  a  few  days. 

This  method  (with  various  slight  modifications  with  regard  to 
dosage)  has  now  been  used  on  a  very  large  scale  in  India,  with 
strikingly  good  results.  The  immunity  lasts  for  at  least  a  year, 
and  probably  decidedly  longer  if  large  doses  of  strong  vaccines 
are  used,  and,  what  is  somewhat  unusual,  it  manifests  itself  more 
in  a  reduction  of  the  incidence  of  the  disease  than  in  the 
case- mortality.  The  value  of  the  method  is  best  seen  from 
statistics  from  isolated  regions  in  which  some  persons  were 
vaccinated  and  others  not,  all  living  under  the  same  conditions. 

26 


4O2  PLAGUE 

Thus,  in  the  tea-plantations  at  Catchar,  in  6,549  persons  who  were 
not  vaccinated,  there  were  198  cases,  with  124  deaths,  whilst  in 
5,778  vaccinated,  there  were  27  cases,  with  14  deaths — i.e.,  the 
incidence  fell  from  3  to  under  0-5  per  cent,  the  case-mortality 
being  62  and  51  per  cent,  respectively.  Numerous  other 
examples  might  be  quoted,  and  the  value  of  the  method  is  now 
proved  to  the  full. 

Plague. 

The  plague  bacillus  produces  a  powerful  endotoxin,  cultures 
killed  by  heat  being  markedly  irritating.  There  is  some  evidence 
that  a  true  exotoxin  may  be  produced,  though  in  small  amounts. 
Filtrates  from  young  cultures  are  devoid  of  toxicity,  whereas 
those  from  older  ones  may  be  fairly  potent.  The  fluid  portion 
of  culture  (in  broth  grown  at  20°  C.  and  kept  well  aerated) 
two  months  old  was  found  by  Markl  to  kill  rats  in  doses  of 
c'i  c.c.  This  might,  of  course,  be  due  to  an  autolysis  of  the  bacilli, 
but  this  seems  improbable  from  the  fact  that  the  toxicity  of  the 
filtrate  is  very  easily  destroyed  by  heat,  whereas  the  endotoxin  is 
thermostable.  These  filtrates  have  slight  immunizing  properties, 
but  the  plague  anti-endotoxin  has  not  been  closely  studied. 

Immunity  appears  to  be  due  to  the  production  of  bacteriolytic 
substances  :  antiplague  serum,  prepared  by  immunizing  horses 
first  with  dead  and  then  with  living  bacilli,  is  powerfully  bacteri- 
cidal. According  to  Wright,  the  plague  bacillus  is  quite  insensible 
to  the  bactericidal  action  of  human  blood,  and  recovery  is  due  to 
opsonization  followed  by  phagocytosis. 

The  agglutination  reaction  is  well  marked  in  artificially 
prepared  immune  serum,  which  may  clump  at  i :  1,000  or  more 
and  may  be  of  use  in  the  identification  of  a  doubtful  bacillus.  It 
s  not  usually  marked,  and  may  be  absent  in  human  cases  of  the 
disease,  and  the  diagnosis  is  most  frequently  made  by  the 
identification  of  the  bacillus  in  fluid  from  a  lesion  or  from  the 
blood  or  sputum.  According  to  Cairns,  the  blood  does  not  usually 
clump  until  the  disease  has  been  in  progress  for  about  a  week. 
The  strength  of  the  reaction  is  not  great,  rarely  rising  above  i  :  50, 
and  is  sometimes  as  low  as  1:3  or  1:5.  The  macroscopic 
method  is  advisable. 

The  curative  treatment  of  the  disease  by  specific  methods 
resolves  itself  into  the  use  of  a  serum,  vaccines  not  having  been 
tried,  as  far  as  I  am  aware.  Several  sera  are  prepared,  but  not  all 


PRACTICAL   APPLICATIONS  403 

have  had  an  extensive  trial.  Yersin's  serum  is  prepared  at  the 
Pasteur  Institute,  the  process  being  to  immunize  horses  for  long 
periods — up  to  a  year  and  a  half — by  weekly  intravenous  injections 
(which  do  not  cause  abscesses,  as  is  the  case  if  the  injections  are 
given  subcutaneously).  For  the  first  three  months  or  so  dead 
bacilli  are  used,  afterwards  living  ones,  and  a  very  high  degree  of 
immunity  is  attained.  The  potency  of  the  serum  is  estimated  by 
finding  the  smallest  amount  which,  given  twenty -four  hours 
previously,  will  save  a  mouse  from  a  lethal  dose  of  living  bacilli : 
this  may  be  as  low  as  0-02  c.c.  Lustig's  serum  is  supposed  to  be 
antitoxic  as  well  as  bactericidal.  It  is  prepared  by  the  immuniza- 
tion of  horses  with  a  "toxin"  prepared  by  dissolving  plague  bacilli 
in  i  per  cent,  caustic  soda  solution,  filtering  and  precipitating  with 
dilute  hydrochloric  acid.  (This  has  also  been  suggested  as  a 
vaccine.)  The  precipitate  is  dissolved  in  0*5  per  cent,  sodium 
carbonate  before  use. 

All  observers  are  not  agreed  as  to  the  efficacy  of  these  sera,  but 
there  is  a  decided  preponderance  of  opinion  in  their  favour. 
Yersin's  serum  is  most  used,  and  is  probably  of  the  greater  value. 
A  most  important  point  in  connection  with  its  use  is  that  large 
doses  are  necessary,  and  those  observers  who  have  not  obtained 
good  results  have  in  some  cases  used  quantities  which  were  far 
too  small.  Cairns  used  Yersin's  serum  in  the  Glasgow  epidemic, 
and  in  severe  cases  gave  150  to  200  c.c.,  part  in  the  region  draining 
into  the  affected  glands  and  part  intravenously.  Choksy,  as  the 
result  of  large  experience,  urges  the  importance  of  a  very  early 
use  of  the  remedy,  and  gives  60  to  100  c.c.  for  adults  and  10  c.c. 
for  infants,  giving  fresh  injections  of  gradually  diminishing  amounts 
every  twenty-four  hours,  until  six  or  eight  have  been  given  in  all — 
150  to  300  c.c.  He  used  Lustig's  serum.  In  any  case  the  effect 
of  serum  is  not  a  great  one,  a  lowering  of  the  case-mortality  by 
about  i  o  to  20  per  cent,  being  apparently  the  utmost  to  be  hoped 
for  at  present.  It  appears,  however,  that  no  other  treatment 
available  is  so  successful. 

The  question  of  the  preventive  treatment  is  much  more  im- 
portant. In  some  cases  the  serum  may  be  used,  and  is  probably 
most  efficacious ;  but  its  effects  are  but  transitory,  and  its  only 
legitimate  use  is  to  tide  the  person  over  the  time  until  vaccination 
can  be  performed  and  active  immunity  acquired. 

Haftkine's  plague  prophylactic  consists  of  a  virulent  broth 
culture  of  the  bacillus,  killed  by  heat  and  preserved  by  the 

26 — 2 


4°4 


PLAGUE 


addition  of  0*5  per  cent,  carbolic  acid.  Cultures  are  made  in 
peptonized  broth  to  which  a  small  amount  of  oil  is  added.  This 
floats  on  the  surface,  and  serves  as  a  point  of  attachment  for  the 
characteristic  "  stalactites."  The  flasks  are  kept  at  the  ordinary 
temperature  (of  Bombay — about  27°  C.)  and  shaken  occasionally, 
to  break  up  the  stalactites.  Incubation  lasts  five  to  six  weeks. 
The  vaccine  is  sterilized  at  65°  C.  for  one  hour.  The  dose  is 
2-5  c.c.  Constitutional  and  local  symptoms  of  moderate  severity, 
and  lasting  for  a  few  days,  are  produced,  but  the  patient  is  as  a 
rule  able  to  follow  his  ordinary  occupation.  The  immunity 
seems  to  be  developed  quite  quickly,  so  that  there  is  no  reason  to 
fear  any  ill-effects  from  the  injections  when  the  patient  is  actually 
exposed  to  plague,  and  perhaps  even  infected.  According  to 
Bannerman,  the  protection  is  developed  in  twenty-four  hours,  and 
asts  about  eighteen  months. 

Of  the  value  of  the  method  there  can  be  no  doubt,  and  statistics, 
both  those  on  a  large  scale  and  those  dealing  with  communities, 
some  of  whom  are  vaccinated  and  some  not,  prove  clearly  that 
the  treatment  lowers  the  likelihood  of  infection,  and  also  the  case- 
mortality.  Thus,  in  twelve  districts  in  the  Punjab  in  which 
plague  was  raging  in  the  winter  of  1902-03  the  following  results 
were  obtained : 


Total. 

Cases. 

Per 
Cent. 

Deaths. 

Per           Case- 
Cent.     Mortality. 

Uninoculated    (average 

l 

population  of  district) 

639,630 

49,433 

77 

29,733 

47        60-  1 

Inoculated    (average 

population  of  district) 

186,797 

3-399 

1-8 

814 

0-4        23-9 

With  regard  to  the  second  group  of  statistics,  the  experience  in 
Umarkadi  Gaol  may  be  quoted,  as  one  out  of  many.  Half  the 
prisoners,  selected  purely  by  chance,  were  inoculated,  and  all 
lived  together  under  exactly  the  same  conditions.  Some  of  each 
group  were  liberated,  and  of  the  remainder  there  were  127  non- 
inoculated,  with  10  cases  and  6  deaths,  and  147  vaccinated,  with 
3  cases  and  no  death. 

The  German  Commission  recommended  the  use  of  vaccines 
prepared  from  two-day-old  agar  cultures,  sterilized  by  heat.  This 
is  more  easily  and  quickly  prepared  than  Haftkine's  fluid. 

The  combined  method  (use  of  vaccine  and  serum)  has   been 


PRACTICAL   APPLICATIONS  405 

recommended  by  Calmette,  by  Besredka,  and  by  Shiga ;  the  last- 
named  obtained  very  good  results  by  its  use  in  an  epidemic  in 
Kobe. 

Anthrax. 

The  nature  of  the  toxin  of  anthrax  is  quite  unknown,  although 
it  has  been  the  subject  of  much  experimental  investigation.  No 
exotoxin  is  formed  in  ordinary  media.  If  coagulable  or  coagulated 
proteids  are  present  in  the  medium,  they  will  be  broken  down  into 
peptones,  etc.,  which  have  some  toxic  action,  but  no  true  toxin  is 
produced.  Some  observers  have  found  that  the  filtrate  from  broth 
cultures  of  anthrax,  though  devoid  of  toxicity,  may  have  some 
immunizing  powers,  a  result  which  we  should  now  attribute  to 
the  presence  of  free  receptors.  The  only  importance  attaching  to 
these  facts  is  that  they  may  explain  the  results  obtained  by  some 
investigators,  who  obtained  albumoses  and  other  bodies  of  very 
feeble  toxicity  from  various  culture  media,  and  considered  them 
to  be  the  true  toxin  because  they  served  to  immunize  animals. 
And,  according  to  Conradi,  there  is  no  evidence  in  favour  of  the 
existence  of  an  endotoxin.  Bacilli  killed  by  various  methods  and 
disintegrated  by  Buchner's  process  yielded  a  non-toxic  fluid. 
The  clinical  nature  of  the  disease  in  some  of  its  manifestations 
(especially  pulmonary  anthrax)  would  rather  lead  us  to  believe 
that  a  powerful  toxin  is  produced,  but  of  this  there  is  not  the 
slightest  shred  of  experimental  verification. 

The  process  of  recovery  and  the  subsequent  immunity  are  also 
very  difficult  to  understand.  Local  immunity  is  very  marked, 
the  skin  being  highly  resistant  in  comparison  with  the  lungs,  an 
infection  of  which  region  forms  one  of  the  most  rapid  and  intract- 
able diseases  known  in  man.  There  are  very  marked  differences 
with  regard  to  the  immunity  of  different  animals.  The  fowl  is 
highly  immune,  as  are  cold-blooded  animals.  The  rat  and  dog 
are  partially  immune,  whereas  sheep,  cattle,  and  the  small  animals 
of  the  laboratory  are  very  susceptible. 

It  is  especially  noteworthy  in  the  case  of  anthrax  that  the 
presence  of  bactericidal  substances  in  the  blood  is  no  indication 
whatever  as  to  the  degree  of  immunity*  The  serum  of  the  rabbit, 
a  highly  susceptible  animal,  has  an  extremely  powerful  bactericidal 
effect,  whereas  that  of  the  dog  and  rat  have  but  little.  The 
classical  Pfeiffer's  phenomenon  is  not  seen  in  the  case  of  this 
bacillus,  but  the  altered  bacteria  may  be  readily  recognized  from 


406  ANTHRAX 

the  fact  that  they  fail  to  stain  by  Gram's  method.  This  change 
is  brought  about  very  quickly  by  a  suitable  serum,  the  change 
being  often  complete  in  ten  minutes  at  37°  C. 

There  have  been  numerous  attempts  to  explain  the  apparent 
anomalies  of  the  reaction  in  question.  Bail  found  that  dog  serum 
(normally  a  good  culture  medium  for  the  anthrax  bacillus) 
becomes  highly  bactericidal  after  the  addition  of  a  small  amount 
of  rabbit  serum,  even  when  this  is  only  present  in  amount  so 
small  that  it  is  devoid  of  bactericidal  action  per  se.  This  appears 
to  be  due  to  the  presence  of  immune  body  in  the  dog's  blood,  but 
no  complement.  If  the  action  of  the  rabbit's  serum  is  due  to  the 
presence  of  complement,  this  must  be  thermostable,  for  the  effect 
is  not  annulled  by  heating  to  50°  C.  Bail  and  Petterson  found 
that  many  other  sera  could  be  reactivated  with  rabbit  serum 
(man,  ox,  calf,  pig,  etc.),  and  that  extracts  of  leucocytes  or  of 
organs  (liver,  bone-marrow)  might  be  equally  effective.  Malvoz 
also  investigated  the  presence  of  immune  body  by  means  of  the 
Bordet-Gengou  reaction  (absorption  of  complement),  and  found 
that  the  amount  in  the  serum  was  some  index  as  to  the  degree  of 
immunity.  Thus  the  blood  of  the  ox  and  guinea-pig  contain 
none,  as  is  the  case  with  the  newly-born  puppy,  an  animal 
susceptible  to  anthrax,  whereas  the  adult  dog  contains  a  large 
amount.  Remy  has  also  studied  the  question  of  the  reactivation 
of  sera  of  various  species  by  complements  from  others,  and 
notably  that  of  the  fowl.  Thus  the  serum  of  the  white  rat  (an 
immune  animal)  contains  an  immune  body,  for  after  heating  to 
55°  C.  it  can  be  reactivated  with  fowl  serum.  On  the  other  hand, 
the  serum  of  the  goat  after  heating  cannot  be  rendered  bacteri- 
cidal in  this  way.  He  holds  that  there  is  an  absolute  concordance 
between  the  bactericidal  power  of  the  blood,  the  presence  of 
immune  body,  and  the  resistance  of  the  animal  to  infection  with 
this  organism. 

Sobernheim  and  others  have  explained  the  susceptibility  of  the 
rabbit  by  supposing  that  the  immune  body  has  a  greater  affinity 
for  the  cells  of  the  animal  than  for  the  anthrax  bacillus,  and  is 
thus  absorbed  and  rendered  useless. 

On  the  other  hand,  Metchnikoff  holds  that  the  immunity  is 
entirely  due  to  phagocytosis,  and  finds  that  the  extent  to  which 
the  bacteria  are  taken  up  by  the  leucocytes  is  proportional  to  the 
degree  of  resisting  power.  Anthrax  bacilli  (and  especially  the 
second  vaccine,  which  forms  a  very  good  emulsion)  are  very 


PRACTICAL  APPLICATIONS  407 

suitable  objects  for  the  study  of  phagocytosis.  They  are  taken  up 
with  great  rapidity,  and  quickly  undergo  solution  within  the 
leucocyte,  first  losing  their  sharp  outline  and  power  of  retaining 
Gram's  stain,  and  disappearing  altogether  in  ten  minutes  or  less. 
This  makes  the  study  of  the  opsonic  index  a  matter  of  some 
difficulty,  which  can  be  overcome  by  using  isolated  spores  in  test- 
tube  experiments.  When  no  serum  is  used  very  few  bacilli  or 
spores  are  taken  up,  and  before  the  discovery  of  the  opsonins 
Metchnikoff  noted  that  when  rats  are  injected  on  the  one  side 
with  anthrax  bacilli  and  on  the  other  with  the  same  organisms 
mixed  with  blood-serum,  oedema  occurs  only  at  the  former  place, 
and  it  is  from  this  that  generalization  occurs.  Sawtchenko  also 
found  that  when  the  injection  of  the  needle  causes  haemorrhage 
the  rat  survives.  The  very  careful  and  full  researches  of  Metch- 
nikofF  on  the  degree  of  phagocytosis  in  susceptible  and  non- 
susceptible  animals  are  probably  sufficient  to  lead  us  to  believe 
that  the  ingestion  of  the  bacilli  by  the  leucocytes  is  the  all-im- 
portant process  in  the  cure  of  the  disease,  and  the  discovery  of 
the  opsonins  supplies  the  missing  link  necessary  for  us  to  account 
for  all  the  facts  in  a  fairly  satisfactory  manner.  We  can  only 
conclude  that  the  bactericidal  effect  of  the  serum  plays  a  part  of 
comparatively  small  importance  in  combating  the  disease  —  the 
elaborate  researches  of  Bail,  Petterson,  Sobernheim,  etc.,  to  the 
contrary  —  possibly,  but  by  no  means  certainly,  owing  to  the 
absence  of  complement. 

The  facts  of  passive  immunity  are  not  so  fully  explained. 
There  are,  however,  some  reasons  for  thinking  that  the  active 
substance  is  an  opsonin,  perhaps  a  thermostable  one.  Thus 
Sclavo's  serum  (according  to  Cler)  will  render  bacilli  fit  for 
ingestion  after  five  hours'  contact,  and  it  does  not  lose  its  efficiency 
on  keeping.  On  the  other  hand,  the  remarkably  rapid  improve- 
ment sometimes  seen  after  the  -fise  of  Bandi's  serum  rather 


suggests  the  presence  of  an  antitoxin. 

Diagnosis.  —  This  is  made  in  all  cases  by  the  demonstration  of 
the  bacillus. 

Treatment.  —  The  preventive  treatment  is  used  for  animals  only. 
Pasteur's  method  has  already  been  noticed  :  it  has  been  largely 
used,  and  the  results  have,  on  the  whole,  been  good.  The 
mortality  from  the  inoculation  is  about  \  per  cent,  of  all  cases, 
but  in  some  herds  the  number  of  deaths  is  much  higher,  and 
serious  loss  is  caused.  The  immunity  is  supposed  to  last  for  less 


408  ANTHRAX 

than  a  year,  when  a  reinoculation  is  necessary.  The  method  is 
not  free  from  objections,  but  its  use  in  regions  of  France  where 
anthrax  was  very  prevalent  proved  of  enormous  value,  and  areas 
in  which  raising  cattle  and  sheep  was  rapidly  becoming  im- 
possible were  practically  cleared  of  the  disease.  The  weak  point 
of  the  process  is  that  the  immunity  to  infection  through  the 
alimentary  canal,  if  it  exists,  is  extremely  feeble. 

To  remedy  the  defects  of  Pasteur's  system,  Sobernheim  has 
introduced  a  method  of  conferring  mixed  immunity.  An  anti- 
anthrax  serum  and  a  culture  resembling  Pasteur's  second  vaccine 
are  injected  simultaneously  into  different  parts  of  the  body,  and 
no  second  inoculation  is  given.  The  doses  are  5  to  15  c.c.  of  the 
serum  and  0^5  to  i  c.c.  of  culture.  This  method  of  treatment  is 
said  to  be  free  from  danger,  to  protect  against  infection  via  the 
intestinal  tract ;  it  has  also  the  advantage  of  requiring  only  a 
single  visit.  The  serum  is  also  curative. 

Curative  Treatment. — Here  the  use  of  serum  is  indicated.  Sclavo's 
serum  is  most  used  in  this  country.  It  is  obtained  by  immuniz- 
ing the  animals  with  Pasteur's  vaccines,  and  then  by  giving  large 
doses  of  virulent  bacilli  mixed  with  gelatin,  which  seems  to 
prevent  the  formation  of  abscesses.  The  dose  is  20  to  40  c.c., 
repeated  in  twenty-four  hours  if  necessary,  or  four  or  five  doses 
of  20  c.c.  each :  the  first  injection  may  advantageously  be 
intravenous.  It  is  usually  followed  by  improvement  within 
twenty-four  hours,  and  often  causes  sweating  and  a  rise  of 
temperature.  Sobernheim's  serum  is  obtained  by  a  somewhat 
different  method,  and  appears  to  be  equally  efficacious.  The  dose 
recommended  is  20  c.c. 

The  results  of  the  use  of  serum  in  malignant  pustule  (which  is 
not  so  dangerous  a  disease  as  was  once  thought,  even  if  untreated 
by  serum,  the  knife,  cautery,  etc.)  have  been  very  satisfactory: 
there  do  not  seem  to  be  any  observations  on  its  use  in  the 
far  more  serious  woolsorter's  disease  or  pulmonary  anthrax. 
Malignant  pustule  is  also  treated  by  the  use  of  very  hot  fomenta- 
tions, the  idea  being  to  bring  about  the  attenuation  of  the  bacillus. 
There  is  little  doubt  that  vaccines  might  be  used  if  thought 
desirable  in  the  absence  of  serum. 

Diphtheria. 

Diphtheria  presents  a  close  approach  to  our  idea  of  a  disease  the 
immunity  to  which  is  antitoxic,  but  it  is  erroneous  to  imagine  that 


PRACTICAL   APPLICATIONS  409 

the  neutralization  of  the  toxin  or  its  destruction  or  elimination 
constitutes  the  whole  process  of  cure.  There  is  a  little  evidence 
in  favour  of  the  formation  of  bacteriolytic  substances,  though 
experimental  evidence  on  this  point  is  not  unanimous.  Bandi,  it 
is  true,  claimed  to  have  been  able  to  immunize  animals  to  the 
bacilli  themselves,  and  prepared  a  serum  which  was  supposed  to 
have  bactericidal  properties ;  it  has  been  prepared  by  others,  and 
can  be  obtained  commercially.  It  is  supposed  to  be  used  locally, 
either  in  the  form  of  a  powder  or  of  lozenges,  and  is  intended  to 
supplement  the  action  of  antitoxin.  Rist,  however,  failed  to 
immunize  animals  to  the  bodies  of  the  bacilli,  and  though  Lipstein 
was  more  successful,  his  serum  was  apparently  inert  as  a  protective 
agent.  It  contained,  however,  an  agglutinin,  and  the  interesting 
fact  was  noticed  that  it  only  clumps  bacilli  of  the  culture  used 
for  the  injection.  This  is  of  some  interest,  since  the  Klebs-Loffler 
bacillus  has  always  been  looked  upon  as  a  very  definite  bacterial 
species;  the  toxins  it  produces  are  always  neutralized  by  the 
same  antitoxin,  and  though  they  may  be  produced  in  larger  or 
smaller  amounts  and  may  contain  varying  proportions  of  proto- 
toxoids,  etc.  (on  Ehrlich's  theory),  appear  to  be  the  same  substance 
in  all  cases.  These  experiments  would  tend  to  show  that,  though 
the  bacilli  of  various  types  agree  in  their  metabolic  products,  they 
may  differ  in  the  constitution  of  their  protoplasm. 

The  observations  referred  to  previously,  show  clearly  that  the 
process  of  cure  of  the  local  lesion  is  assisted  by  the  produc- 
tion of  an  opsonin.  And  there  is  every  reason  to  believe  that 
it  is  by  phagocytosis  that  the  bacilli  are  combated,  bacteriolysis 
being  very  doubtful  and  of  comparatively  small  importance. 
The  cure  of  the  disease  therefore  is  accomplished  partly  by  one 
or  more  of  the  methods  discussed  in  Chapter  VI.,  and  partly 
by  phagocytosis. 

Diagnosis. — This  is  made  by  the  demonstration  of  the  bacillus. 
If  necessary,  the  opsonic  test  might  be  used,  and  Bordet  and 
Gengou  have  shown  by  their  method  of  fixation  of  complement 
that  "  sensibilatrices  "  circulate  in  the  blood.  These  methods  are 
quite  unnecessary.  The  absolute  recognition  of  diphtheria 
bacillus  in  cultures  can  best  be  made  by  an  application  of  an 
immunity  reaction.  A  pure  culture  in  broth  is  divided  into  two 
parts,  and  each  injected  into  a  guinea-pig.  One  of  the  animals 
receives  a  large  dose  of  antitoxin,  and  should  this  remain  unaffected 
whilst  the  other  dies,  the  culture  is  certainly  diphtheria.  The 


410  TETANUS 

method  is  usually  only  required  in  cases  where  a  healthy  person 
contains  diphtheroid  bacilli  in  his  mouth,  nose,  skin,  etc.,  and 
considerations  of  public  health  render  a  determination  of  their 
exact  nature  necessary. 

Treatment. — This  consists  in  the  early  use  of  antitoxin  and  the 
treatment  of  the  local  lesion  with  antiseptics,  and  the  only  question 
of  importance  concerns  the  dosage  of  the  former  remedy.  As  a 
rule,  4,000  to  8,000  units  should  be  given  at  once,  and  a  second 
injection  at  the  end  of  twelve  or  twenty-four  hours  ;  subsequent 
doses  are  given  if  required.  Unless  a  case  is  seen  very  early,  a 
part  at  least  of  the  first  dose  may  be  given  intravenously,  and 
this  is  always  advisable  in  severe  cases  not  seen  until  the  disease 
has  been  present  for  two  or  three  days.  Larger  doses  may  be 
given,  but  are  of  doubtful  advantage ;  a  smaller  amount  should 
not  be  given,  except  perhaps  in  mild  cases. 

The  sole  preventive  treatment  in  actual  use  consists  in  the  use 
of  comparatively  small  doses  of  antitoxin.  The  protection  which 
is  conferred  is  usually  a  strong  one,  but  exceptions  have  been 
recorded.  It  lasts  about  a  month.  Essays  in  vaccination  have 
been  made,  but  not  on  a  large  scale. 

Tetanus. 

The  pathology  of  tetanus  is  akin  to  that  of  diphtheria  in  that  it 
is  a  local  disease  with  remote  symptoms  due  to  the  action  of  a 
soluble  exotoxin  on  distant  structures.  It  differs  from  diphtheria 
mainly  in  two  points  :  the  bacilli  are  strictly  localized  to  the  region 
inoculated  and  the  immediate  neighbourhood,  and  the  toxin,  which 
acts  entirely  on  the  central  nervous  system,  reaches  it  entirely,  or 
almost  so,  by  ascending  the  nerves  from  the  region  in  which 
infection  occurs,  and  not  by  circulating  in  the  blood-stream.  This, 
at  least,  is  the  usual  course  of  events,  and  when,  as  occasionally 
happens,  the  toxin  actually  gains  access  to  the  blood,  it  seems 
likely  that  even  then  it  does  not  act  on  the  brain  direct,  but  enters 
the  peripheral  nerves  at  their  distal  endings  and  then  ascends 
them  to  their  origin. 

The  diagnosis  is  made  entirely  by  the  recognition  of  the 
organism  in  the  wound,  no  agglutination  or  other  tests  being  used. 
If  (as  usually  happens)  the  culture  obtained  from  the  w?ound  is 
impure,  it  is  divided  into  two  parts,  the  one  of  which  is  injected 
alone,  the  other  in  conjunction  with  tetanus  antitoxin.  If  no 
other  pathogenic  bacteria  are  present  the  animal  that  has  received 


PRACTICAL   APPLICATIONS  4! I 

the  mixture  will  survive,  whilst  the  other  will  develop  tetanic 
symptoms  and  die.  Even  if  other  pathogenic  bacteria  are  present 
the  indications  are  usually  clear,  since  spasms  will  commonly 
develop  (in  the  animal  which  has  received  no  antitoxin)  before  the 
lethal  issue.  It  is  best  to  use  a  broth  culture  for  this  test,  so  that 
there  may  be  a  good  development  of  toxins. 

The  nature  of  the  toxins  of  tetanus  have  been  already  mentioned. 
There  are  two,  both  exotoxins — the  real  poison,  tetanospasmin,  and 
tetanolysin.  Tetanospasmin  is  readily  prepared  by  cultivation  of 
the  organism  in  pure  culture  in  almost  any  medium  under  anaerobic 
conditions.  It  is  even  more  fragile  than  diphtheria  toxin,  being 
rapidly  rendered  inert  in  a  few  days  if  exposed  to  air  at  ordinary 
temperatures.  It  is  destroyed  in  eight  to  eighteen  hours  by 
sunlight,  by  a  temperature  of  55°  C.  in  one  and  a  half  hours,  and  by 
exposure  to  agents  such  as  alcohol,  potassium  permanganate,  and 
trichloride  of  iodine.  It  can  be  preserved  by  means  of  dilute 
carbolic  acid  (0-6  per  cent.)  or  chloroform  without  much  loss. 
Inert  solutions  have  in  general  powerful  immunizing  properties, 
the  toxin  being  converted  into  toxoids,  and  not  absolutely 
destroyed. 

It  can  be  prepared  so  as  not  to  give  the  reactions  for  proteid, 
and  is  formed  when  the  bacillus  is  grown  on  Uschinsky's  proteid- 
free  medium.  Its  potency  is  enormous.  Thus  Vaillard  prepared 
a  toxin  of  which  the  lethal  dose  for  a  guinea-pig  was  0*001  c.c., 
containing  about  0*000025  gramme  of  solid  matter,  only  a  small 
portion  of  which  was  pure  toxin.  Brieger  and  Cohn  calculated 
that  the  lethal  dose  of  an  (impure)  toxin  for  a  man  was  0-00023 
gramme. 

The  effect  of  tetanus  toxin  is  manifested  almost  solely  on 
the  central  nervous  system,  and  the  post-mortem  lesions  are 
practically  confined  to  the  ganglionic  cells,  especially  of  the 
anterior  cornua.  It  appears  probable  that  there  is  no  direct 
action  on  the  nerves  themselves,  but  the  toxin,  like  the  virus  of 
rabies,  reaches  the  central  nervous  system  mainly,  if  not  entirely, 
by  ascending  the  nerves  leading  from  the  area  of  inoculation. 
According  to  Meyer  and  Ransom,  toxin  which  gains  access  to  the 
blood  only  affects  the  brain  by  entering  the  peripheral  nerves  via 
the  nerve  endings,  especially  the  end-plates,  but  this  is  not 
universally  accepted.  As  in  the  case  of  rabies,  the  richer  the  area 
of  inoculation  in  nerves,  the  more  powerful  the  action  of  the  toxin 
and  the  shorter  the  period  of  incubation.  The  brain  and  cord  are 


412  TETANUS 

the  most  susceptible  regions,  the  peripheral  nerves  next,  then 
regions  with  an  abundant  nerve  supply,  such  as  the  face ;  and 
lastly,  regions  poorly  supplied,  such  as  the  subcutaneous  and 
peritoneal  tissues.  The  incubation  period  of  tetanus  is  thus  seen 
to  be  composed  of  :  (i)  the  time  necessary  for  the  production  of  the 
toxin  in  the  tissues ;  (2)  for  its  ascent  of  the  nerves  to  the  brain 
being  longer,  other  things  being  equal,  if  infection  takes  place  at 
a  long  distance  therefrom  ;  and  (3)  the  latent  period  which  elapses 
after  the  toxin  has  united  with  the  ganglion  cells  of  the  central 
nervous  system,  and  before  the  development  of  symptoms — i.e., that 
in  which  the  enzyme-like  action  of  the  zymophore  group  is  being 
gradually  exerted  on  the  protoplasm.  The  fixation  of  tetanus 
toxin  in  the  system  is  extremely  rapid:  in  rabbits  it  may  dis- 
appear entirely  from  the  blood  in  one  minute,  whilst  in  other 
susceptible  animals  it  circulates  for  slightly  longer  periods.  The 
importance  of  this  arises  from  the  fact  that  toxin  which  has  once 
entered  the  nerves  is  thereby  shielded  from  the  action  of  antitoxin. 
The  dose  of  antitoxin  necessary  to  save  the  life  of  an  animal  which 
has  received  a  few  lethal  doses  of  toxin  rises  enormously  if  the 
injection  of  the  former  is  delayed  more  than  a  few  minutes. 

Tetanolysin  is  even  more  fragile  than  tetanospasmin,  being 
converted  into  toxoids  in  a  few  hours  at  the  room  temperature. 
It  can  be  preserved  in  a  dry  state.  The  role  which  it  plays  in 
natural  infections,  if  any,  is  unknown. 

As  regards  immunity,  there  is  but  little  to  add  to  what  has  been 
discussed  previously.  The  bacilli  are  not  powerful  parasites, 
being  readily  ingested  by  the  leucocytes,  and  destroyed  if  the 
conditions  are  favourable  for  phagocytosis.  In  most  of  the  cases 
which  develop  tetanus  there  is  a  contused  or  lacerated  wound, 
with  much  killed  and  bruised  tissues  and  an  abundant  con- 
comitant infection  with  other  bacteria,  which  still  further  paralyze 
the  natural  resistance  of  the  part.  These  organisms  may  have 
an  additional  influence  in  securing  a  condition  of  anaerobiosis  : 
tetanus  bacilli  grown  in  symbiosis  with  certain  other  bacteria 
which  have  powerful  oxygen-absorbing  properties  will  develop 
vigorously,  and  develop  toxin  in  spite  of  the  free  access  of  air. 
No  observations  with  regard  to  the  opsonic  index  in  tetanus 
appear  to  have  been  recorded.  The  question  of  immunity  to 
tetanus  toxin  has  been  dealt  with  already,  but  we  may  add  that 
in  all  probability  much  of  the  toxin  is  destroyed  in  loco  by  the 
unspecific  action  of  the  peptic  enzyme  formed  by  the  leucocytes 


PRACTICAL    APPLICATIONS  413 

Antitoxin  is  rarely,  if  ever,  found  in  human  patients  who  have 
survived  an  attack  of  the  disease. 

Treatment. — The  main  question,  of  course,  concerns  the  use  of 
antitoxin,  and  two  general  rules  may  be  laid  down  :  (i)  It  is  of 
great  value  as  a  prophylactic  agent,  and  (2)  it  is  of  some  value  in 
chronic  tetanus— i.e.,  the  form  with  mild  symptoms  developing  after 
a  long  period  of  incubation. 

Its  preventive  application  is  indicated  in  the  treatment  of  all 
wounds  which  experience  has  shown  to  be  followed  by  tetanus 
— i.e.,  lacerated  and  contused  wounds,  especially  if  contaminated 
with  garden  soil,  road  debris,  etc.  Gunshot  wounds  are  especially 
dangerous,  and  tetanus  is  usually  extremely  prevalent  in  warfare. 
It  is,  of  course,  somewhat  difficult  to  estimate  precisely  the  value 
of  the  treatment,  inasmuch  as  tetanus  is  not  a  common  disease  ; 
but  experience  derived  from  horses,  which  animals  are  extremely 
prone  to  it,  is  more  conclusive.  In  some  veterinary  practices  it 
was  so  common  as  to  counterindicate  any  operative  measure,  and 
has  now  been  completely  eradicated.  The  duration  of  the 
immunity  conferred  by  a  single  dose  is  about  three  weeks,  and 
in  the  prophylactic  treatment  of  wounds,  whether  accidental  or 
due  to  operation,  two  doses  should  be  given,  at  intervals  of  ten 
to  fourteen  days.  The  prophylactic  treatment  of  dirty  wounds  by 
means  of  antitoxin  is  now  a  routine  method  in  several  Continental 
clinics,  and,  as  far  as  I  am  aware,  there  has  been  no  case 
recorded  in  which  it  has  been  followed  by  the  development  of  the 
disease,  excepting  those  in  which  the  injection  has  been  given 
some  days  after  the  injury,  when  the  toxin  has  already  gained 
access  to  the  nerves.  One  case  (under  Mr.  Lenthal  Cheatle) 
from  which  I  isolated  a  bacillus  identical  in  cultural  and  morpho- 
logical characters  with  that  of  tetanus,  and  in  which  the  organisms 
occurred  in  great  abundance,  was  treated  with  antitoxin  at  the 
outset,  and  healed  without  a  symptom  of  the  disease  :  the  culture 
was  unfortunately  not  tested  by  inoculation. 

Calmette  prepares  a  powder  of  dry  antitetanus  serum  to  be  used 
as  a  dressing  for  wounds,  but  its  use  is  very  doubtful.  Anti- 
toxin is  a  good  culture  medium  for  bacteria,  and  unless  the  wound 
is  fairly  clean  may  decompose  and  become  offensive.  The  most 
scrupulous  antiseptic  technique  should  be  adopted,  and  it  seems 
probable  that  the  dry  dressing  presents  no  advantage  over  the 
subcutaneous  administration  of  the  serum  when  this  is  done. 

The  doses  should  be  5  to  10  c.c.  for  a  man,  and  10  to  20  c.c.  for 


4*4  TETANUS 

a  horse.  The  best  method  of  standardization  is  that  of  Roux,  who 
determines  the  amount  of  serum  necessary  to  protect  a  guinea-pig 
weighing  500  grammes  against  ten  lethal  doses  of  toxin.  The  result 
is  expressed  in  terms  of  the  weight  of  guinea-pig  protected  against 
one  lethal  dose  of  antitoxin  by  i  c.c.  of  serum — e.g.,  if  ^  c.c.  pro- 
tected a  guinea-pig  weighing  500  grammes  against  ten  lethal  doses, 
the  potency  would  be  50,000.  A  potency  of  1,000,000  is  the  least 
that  should  be  employed. 

The  use  of  tetanus  antitoxin  in  the  developed  disease  is  less 
satisfactory,  a  fact  readily  explicable  nowr  that  the  pathology  of 
the  disease  is  more  fully  understood.  In  acute  tetanus  it  is 
practically  worthless,  though  a  few  cures  have  been  reported.  In 
many  cases  of  chronic  tetanus  it  is  without  action  ;  in  a  few, 
however,  it  is  decidedly  beneficial,  each  injection  greatly  alle- 
viating the  patient's  suffering.  It  is  always  worthy  of  trial,  but 
it  is  hardly  necessary  to  say  that  the  non-specific  treatment 
should  not  be  neglected.  If  the  patient  has  a  sufficient  degree  of 
immunity  to  resist  the  toxin  which  has  already  gained  access  to 
his  nervous  system,  the  antitoxin  will  be  of  value  in  preventing  any 
more  from  doing  so,  inasmuch  as  it  will  neutralize  it  as  soon  as  it 
is  formed. 

The  doses  should  be  large  — 20  c.c.  or  more  at  first,  and  10  c.c. 
every  day,  or  every  alternate  day,  subsequently.  The  site  of 
inoculation  is  of  some  importance.  The  injections  may  be  given 
subcutaneously  in  a  distant  region,  as  in  the  use  of  diphtheria 
antitoxin ;  but,  in  view  of  the  fact  that  it  takes  an  appreciable 
time  for  it  to  be  absorbed — and  time  is  of  the  utmost  value  if  the 
remedy  is  to  be  of  any  use — it  seems  advisable  to  give  the  first 
dose  either  in  the  region  of  the  wound  or  intravenously. 

Various  methods  have  been  proposed  by  which  the  antitoxin 
can  be  brought  into  closer  relation  with  the  nerve  elements. 
The  intracerebral  injection  has  most  to  recommend  it  on  theoretical 
grounds,  and  several  very  decided  successes  have  been  recorded 
in  severe  cases  of  the  disease.  The  method  is  as  follows  :  A 
small  flap  of  the  scalp  (with  its  base  downwards)  is  reflected  so 
as  to  expose  the  skull  a  little  to  one  side  of  the  middle  line,  and 
just  in  front  of  the  fronto-parietal  suture.  A  small  trephine  hole 
is  made  through  the  skull,  and  an  exploring  needle  is  inserted 
until  the  lateral  ventricle  is  reached,  and  cerebro-spinal  fluid 
escapes  through  the  needle.  Ten  c.c.  or  more  of  the  serum  are  in- 
jected. This- passes  down  the  ventricular  system,  and  bathes  the 


PRACTICAL   APPLICATIONS  415 

respiratory  and  cardiac  centres  at  the  floor  of  the  fourth  ventricle. 
Another  method  is  to  inject  small  quantities  of  the  fluid  directly 
into  the  spinal  cord  by  means  of  a  needle  introduced  between  the 
sixth  and  seventh  cervical  vertebrae.  This  procedure  would 
appear  to  be  dangerous,  but  this  is  said  not  to  be  the  case. 
Lastly,  the  simplest  method  of  all  is  to  perform  lumbar  puncture, 
draw  off  some  of  the  cerebro-spinal  fluid,  and  replace  it  with 
serum,  just  as  in  the  process  of  spinal  anaesthesia. 

Ransom  and  Meyer  have  advocated  the  direct  application  of 
antitoxin  to  the  nerves  supplying  the  region  in  which  the  wound 
is  situated,  the  idea  being,  of  course,  to  intercept  any  further 
access  of  toxin  to  the  brain  and  cord.  The  nerves  are  exposed 
by  operation  as  near  to  their  origin  as  possible,  and  infiltrated 
with  serum  by  means  of  a  hypodermic  syringe. 

Analogy  with  other  diseases  would  fully  justify  the  use  of 
vaccine  in  chronic  tetanus.  Its  preparation  would  present  some 
difficulties,  owing  to  the  heat-resisting  power  of  the  spores. 

Syphilis. 

Little  is  known  definitely  concerning  the  mode  of  cure  or  of  the 
type  of  immunity  of  syphilis.  It  used  to  be  regarded  as  one  of 
the  diseases  which  are  followed  by  practically  complete  immunity 
of  long  duration,  but  .Neisser  has  brought  forward  some  evidence 
for  thinking  that  this  is  not  the  case,  and  that  it  only  lasts  as 
long  as  the  disease  itself — i.e.,  as  soon  as  it  is  completely  eradicated 
the  patient  is  again  susceptible.  Nothing  is  known  as  to  the 
toxins  of  syphilis,  and,  as  regards  the  method  of  cure,  the  only 
point  worth  mentioning  is  the  fact  that  spirochaetes  which  have 
been  ingested  by  the  leucocytes  can  be  ma^e  out  occasionally. 
They  stain  badly,  and  are  doubtless  on  the  way  to  complete 
absorption.  The  fact  that  the  organism  cannot  be  obtained  in 
pure  culture  renders  researches  with  regard  to  the  opsonic  and 
bacteriolytic  action  of  the  serum  very  difficult.  Indirect  researches 
by  means  of  the  deviation  of  complement  —  constituting  the 
Wassermann  reaction,  a  special  method  of  application  of  the 
Bordet-Gengou  reaction — have  led  to  results  of  great  interest 
which  have  recently  attracted  much  attention. 

The  first  necessity  was,  of  course,  the  preparation  of  an  antigen, 
and  for  this  purpose  Wassermann  made  use  of  the  internal  organs 
of  a  syphilitic  foetus,  which  were  swarming  with  spirochaetes.  In 


416  SYPHILIS 

its  main  outlines  the  technique  is  exactly  the  same  as  that  already 
described.  The  serum  to  be  tested  is  heated,  to  remove  comple- 
ment, and  diluted  with  sterile  normal  saline  solution.  A  dilution 
of  i  :  20  or  i  :  40  is  generally  correct,  but  the  point  may  be  deter- 
mined by  preliminary  tests  with  a  known  syphilitic  serum  ;  and  in 
any  case  it  is  an  advantage  to  perform  a  series  of  tests  with 
different  dilutions,  so  that  a  rough  idea  of  the  amount  of  antibody 
present  in  the  serum  may  be  obtained.  This  is  mixed  with  an 
extract  of  the  syphilitic  organ  (antigen),  and  some  fresh  guinea- 
pig  serum  (complement)  added.  The  proportions  may  be  i  c.c. 
of  diluted  serum,  0*1  or  0-2  c.c.  of  organ  extract,  and  0*2  c.c.  of  fresh 
serum.  The  whole  is  incubated  for  one  hour,  at  the  end  of  which 
time  all  the  complement  will  be  removed  from  the  fluid  if 
syphilitic  antibody  is  present.  Next,  corpuscles  (e.g.,  of  a  sheep  or 
pigeon)  are  added,  together  with  heated  serum  from  a  rabbit 
which  has  been  injected  with  the  corpuscles  in  question  ;  or  the 
corpuscles  may  previously  be  sensitized  with  the  inactivated 
serum,  washed,  and  then  added.  The  whole  mixture  is  then 
incubated  for  two  hours,  with  occasional  stirring  or  shaking, 
and  kept  some  hours  in  the  ice-chest.  A  positive  reaction  is 
shown  by  the  absence  of  haemolysis.  Control  tests  are  also 
advisable — e.g.,  the  corpuscles  must  be  completely  dissolved  by 
the  heated  immune  serum  and  the  guinea-pig's  serum  if  the  other 
two  ingredients  are  not  added,  and  there  should  be  no  haemolysis 
if  all  the  substances  except  the  guinea-pig's  serum  are  used. 

Ledingham  and  Hartoch  have  shown  independently  that 
opsonin  is  absorbed  as  well  as  complement,  and  this  fact  may 
be  used  as  a  test  of  the  presence  of  the  reaction.  In  this  case  the 
first  part  of  the  test  is  performed  as  beforehand  the  fluid  used  as 
the  serum  in  an  opsonin  estimation,  using  staphylococci  or  any 
other  organism,  and  using  as  a  control  guinea-pig  serum  diluted 
with  normal  saline  to  the  same  extent  as  it  was  in  the  mixture  of 
organ  extract,  human  serum,  and  guinea-pig  serum.  In  a  positive 
reaction  the  phagocytic  index  in  the  first  preparation  will  be 
much  below  that  in  the  second  ;  in  a  negative  one  they  will  be 
equal. 

The  exact  value  of  the  test  is  not  yet  quite  definitely  settled. 
It  is  very  rarely  present  in  health,  and  not  common  in  diseases 
other  than  syphilis  ;  but  it  does  occur,  especially  in  diseases  which 
(like  syphilis)  are  due  to  animal  parasites,  such  as  malaria  or 
trypanosomiasis,  and  is  not  uncommon  in  leprosy  and  scarlet 


PRACTICAL  APPLICATIONS  417 

fever.  It  is  rarer  in  other  diseases,  but  isolated  examples  have 
been  met  with  in  systematic  investigations  in  a  great  many 
maladies  ;  but  here  it  is  the  exception,  whereas  in  syphilis  it  is 
the  rule.  In  primary  and  secondary  cases  it  occurs  in  90  per 
cent,  or  more,  and  is  present  in  the  majority  of  patients  suffering 
from  tertiary  syphilis  and  "  metasyphilitic  "  affections.  It  is  very 
frequently  found  in  the  cerebro-spinal  fluid  of  general  paralytics 
(80  per  cent,  90  per  cent.,  or  more),  even  when  it  is  absent  from 
the  blood.  It  is  not  so  common  in  tabes,  and  is  extremely  rare 
(if  it  ever  occurs)  in  the  cerebro-spinal  fluid  in  non-syphilitic 
diseases,  with  the  curious  exception  of  scarlet  fever,  in  which  it 
is  almost  constant. 

So  far  there  is  no  theoretical  difficulty  in  the  interpretation  of 
the  phenomenon,  but  a  new  fact  discovered  by  Landsteiner, 
Miiller  and  Potzl  seems  to  show  that  the  reaction  is  of  a  nature 
entirely  different  from  the  ordinary  Bordet-Gengou  phenomenon. 
They  found  that  an  alcoholic  extract  of  a  normal  organ  (e.g.,  of  a 
guinea-pig's  heart  muscle)  might  be  used  instead  of  a  tissue  rich 
in  spirochsetes  ;  and  further  researches  have  shown  that  the  lipoid 
substances  isolated  therefrom,  or  even  comparatively  simple 
substances,  as  lecithin  and  taurocholate  and  glycocholate  of  soda 
(Levaditi  and  Yamanouchi)  give  the  reaction,  although  apparently 
not  so  frequently,  as  when  an  extract  from  a  syphilitic  organ  is  used. 
The  "antigen"  is  soluble  in  hot  alcohol,  and  this  fact  alone  removes 
it  from  the  group  of  true  antigens,  which,  as  we  have  seen,  are 
apparently  all  proteid  in  nature.  According  to  Levaditi,  the  sub- 
stance occurring  in  the  blood  or  cerebro-spinal  fluid  is  not  an  anti- 
body at  all,  but  either  lipoid  substances  or  salts,  or  the  two  in 
combination,  and  they  are  set  free  when  tissues  are  broken  down  in 
a  certain  way,  which  occurs  most  frequently  in  syphilis,  but  may 
take  place  in  other  diseases.  Under  ordinary  circumstances  they 
are  present  in  a  colloid  state/but  form  a  precipitate  with  the  lecithin 
and  allied  substances  extracted  from  normal  organs  by  hot  alcohol, 
and  to  this  precipitate  the  complement  attaches  itself.  According 
to  Forges,  the  serum  of  syphilitics  has  the  power  of  precipitating  an 
emulsion  of  lecithin  (0*5  gramme,  shaken  up  with  0*5  per  cent, 
solution  of  carbolic  acid  in  normal  saline)  when  mixed  therewith 
in  equal  parts.  This  he  proposed  as  a  test  for  syphilis,  and  Nobl 
and  Arzt  found  it  successful  in  80  per  cent,  of  cases.  Subsequently, 
Forges  replaced  the  lecithin  (which  as  usually  bought  is  not  con- 
stant in  composition)  by  a  recently  prepared  I  per  cent,  solution 

2? 


418  RABIES 

of  glycocholate  of  soda.  A  mixture  of  this  with  an  equal  amount 
of  serum  is  incubated  for  five  hours,  and  observed  after  it  has 
stood  sixteen  to  twenty  hours  at  the  room  temperature.  The 
precipitate  is  specially  obvious  near  the  surface. 

Fornet,  Schereschewsky,  Eisenzimmer,  and  Rosenfeld  find 
that  the  sera  of  syphilitics  in  the  early  stages  of  the  disease 
contain  a  precipitogen  which  forms  an  insoluble  compound  with 
a  substance  or  precipitin  present  in  the  serum  of  tabetics  or 
general  paralytics.  When  the  one  is  floated  on  the  other,  a 
characteristic  ring  appears  at  the  area  of  contact.  They  say  that 
normal  serum  rarely  contains  the  precipitin,  but  not  the 
precipitogen.  What  relation  this  has  to  any  immunity  reaction 
is  unknown. 

Rabies. 

The  actual  causal  agent  of  rabies  is  not  yet  definitely  ascertained. 
The  peculiar  structures  known  as  the  corpuscles  of  Negri  which 
occur  in  the  brain,  and  especially  in  the  hippocampus  major,  of 
rabid  animals  appear  to  be  quite  characteristic  of  the  condition, 
and  may  possibly  be  the  actual  parasite,  although  this  is  not  yet 
universally  accepted.  It  seems,  however,  fairly  certain  that  their 
recognition  constitutes  a  sufficient  proof  of  the  presence  of  the 
disease ;  and  this  is  of  great  importance  in  view  of  the  necessity 
for  the  early  commencement  of  the  treatment,  which  is  entirely 
preventive,  and  not  curative.  If  the  dog  by  which  the  patient  has 
been  bitten  is  forthcoming,  the  corpuscles  of  Negri  can  be  demon- 
strated in  a  short  time  by  simple  methods,  and  the  need  for 
Pasteur's  treatment  ascertained  ;  apart  from  this  the  only  method 
is  by  animal  inoculation,  an  emulsion  of  brain  substance  being 
injected  into  the  brains  of  rabbits  after  trephining. 

Rabies  presents  one  of  the  most  striking  examples  of  local 
immunity ;  the  action  of  the  virus  is  manifested  almost  entirely 
on  the  central  nervous  system,  and  in  whatever  part  of  the  body 
the  inoculation  is  made  the  effects  are  only  caused  when  it  has 
reached  the  brain  and  spinal  cord  ;  and  in  doing  so  it  does  not 
gain  access  to  the  blood,  but  ascends  the  peripheral  nerves. 
Hence  the  central  nervous  system  is  extremely  susceptible  to 
injection,  and  the  other  tissues  in  proportion  to  their  richness  in 
nerves.  Subcutaneous  (unless  into  a  region  like  the  paw),  intra- 
venous, or  intraperitoneal  injections  only  convey  the  disease  if  a 
large  amount  of  extremely  potent  virus  is  used.  Hence  it  seems 


PRACTICAL    APPLICATIONS  419 

reasonable  to  suppose  that  there  is  a  fair  amount  of  immunity 
inherent  in  all  the  tissues  except  in  the  nervous  structures,  and 
that  the  living  virus  deposited  elsewhere  may  be  entirely  destroyed 
by  bacteriolysis  or  phagocytosis. 

We  have  already  glanced  briefly  at  Pasteur's  earlier  work  on 
antirabic  inoculations,  and  the  method  by  which  immunity  is 
produced.  Numerous  modifications  of  the  process  have  been 
introduced  since  Pasteur's  time.  Thus  Hogyes  of  Budapest 
makes  use  of  fully  virulent  cords,  but  given  in  extremely  small 
doses ;  and  there  is  some  reason  to  think  that  his  process  does 
not  really  differ  from  that  of  Pasteur,  and  that  in  drying  the  cords 
the  virus  is  gradually  destroyed  and  not  really  attenuated,  so  that 
a  dose  of  a  fourteen-day  cord  really  contains  a  small  trace  of 
virus  of  full  virulence.  A  true  vaccine — i.e.,  a  virus  of  mitigated 
virulence— can  be  obtained  by  passage  through  monkeys  or  birds. 
Further,  though  the  fixed  virus  is  so  potent  for  rabbits,  it  is  quite 
possible  that  its  virulence  for  man  is  slight  or  nil.  Nitsch  was  so 
sure  of  this  that  he  injected  4  to  5  milligrammes  of  the  fresh  cord 
subcutaneously  unto  himself  (in  the  abdominal  region,  a  part 
comparatively  poor  in  nerves)  without  evil  results. 

Another  method,  introduced  by  Marie,  consists  in  the  use  of  injec- 
tions of  a  mixture  of  virus  and  serum  from  an  immunized  animal. 
This  serum  is  prepared  in  a  variety  of  ways,  the  simplest  being  to 
give  the  virus  intravenously.  The  animal  usually  employed  is  the 
sheep,  and  the  injection  consists  of  rabid  brains,  hcatcclf  up  into  a 
fine  emulsion  with  normal  saline  solution,  and  filtered  through 
linen.  The  serum  prepared  from  animals  treated  in  this  way 
possesses  powerful  ancirabic  properties :  when  mixed  with  a 
potent  virus  it  removes  entirely  all  harmful  properties,  so  that  it 
is  quite  innocuous  even  on  intracerebral  injection.  It  can  be 
titrated  against  an  emulsion  of  fixed  virus  of  definite  strength, 
and  by  appropriate  treatment  a  very  potent  serum  can  be  obtained. 
It  is  apparently  quite  useless  in  the  treatment  of  the  developed 
disease  or  of  an  infected  animal,  even  before  the  development  of 
symptoms.  If  it  is  mixed  in  excess  with  fixed  virus  and  injected 
into  animals  these  do  not  develop  rabies  ;  on  the  other  hand,  but 
little  immunity  is  produced,  and  this  is  supposed  to  be  due  to  the 
fact  that  the  virus  is  so  quickly  absorbed  that  it  does  not  act  as 
an  antigen.  But  if  the  mixture  be  allowed  to  stand  for  some 
time,  and  the  virus  then  recovered  by  centrifugalization  and 
washing  with  normal  saline  solution,  the  clot  thus  obtained  has 

27—2 


42O  RABIES 

powerful  immunizing  properties.  In  the  preventive  treatment  of 
rabies  on  Marie's  system  the  fresh  fixed  virus,  made  into  a  fine 
emulsion  with  normal  saline  solution,  is  partially  neutralized  with 
immune  serum,  and  a  dose  of  6  c.c.  (2  c.c.  of  i  :  10  emulsion  of 
virus  and  4  c.c  of  serum)  is  given  in  two  places  under  the  skin  of 
the  abdomen.  This  is  done  for  four  days,  and  then  injections  of 
dried  cord,  beginning  at  that  of  the  sixth  day,  are  commenced. 

Other  methods  involve  the  use  of  heat,  of  chemical  methods 
(e.g.,  partial  digestion  with  gastric  juice,  as  practised  by  Centanni), 
in  order  to  bring  about  attenuation  or  partial  destruction  of  the 
virus. 

Whatever  the  method,  it  appears  necessary  that  the  patient 
should  undergo  a  course  of  active  immunization,  various  causes 
(e.g.,  the  long  incubation  period  and  the  localization  of  the  virus 
in  the  nerves)  rendering  passive  immunity  an  unsafe  method  of 
protection. 

Of  the  value  of  the  process  there  cannot  be  the  slightest  doubt. 

The  incidence  of  hydrophobia  after  the  bite  of  a  rabid  animal 
is  variously  estimated,  the  figures  usually  given  being  about 
15  per  cent,  in  the  case  of  dog-bites,  and  40  to  80  per  cent, 
in  bites  from  wolves.  The  probability  of  the  patient's  developing 
the  disease  depends  on  the  severity  of  the  bite,  its  position 
(i.e.,  whether  in  regions  rich  in  nerves  or  the  reverse),  and 
on  whether  the  bite  is  through  the  clothing,  so  that  some  of 
the  virus  is  wiped  from  the  teeth.  In  the  twenty-two  years  (down  to 
1907  inclusive,  the  last  year  of  which  the  figures  are  available), 
^0,359  patients  have  been  treated  at  the  Pasteur  Institute  in 
Paris,  with  126  deaths — a  death-rate  of  0-31  per  cent.  (The 
patients  dying  within  fifteen  days  of  the  commencement  of  the 
treatment — a  small  number — are  excluded  from  the  figures,  since 
in  them  the  disease  was  too  far  advanced  for  a  preventive  treat- 
ment to  be  of  value.) 


BIBLIOGRAPHY 

[/4  complete  bibliography  of  the  subject  being  obviously  impossible  in  any  reasonable 
space,  an  attempt  has  been  made  to  include  important  articles,  and  especially 
those  referred  to  in  the  text,  and  articles  dealing  with  the  subjects  in  a  complete 
manner,  especially  those  with  a  good  account  of  the  literature.'} 

CHAPTER  I 

GOOD  accounts  of  the  general  phenomena  of  immunity  may  be  found  in 
Metchnikoff's  "  L'Immunite  dans  les  Maladies  Infectieuses  "  (English  trans- 
lation by  F.  G.  Binnie,  University  Press,  Cambridge)  ;  Ricketts'  "  Infection, 
Immunity,  and  Serum  Therapy  "  (American  Medical  Associated  Press, 
Chicago)  ;  in  Clifford  Allbutt's  "  System  of  Medicine,"  vol.  ii.,  part  i.  ;  in 
Muir  and  Ritchie's  "  Bacteriology."  Also  Levaditi's  "  La  Nutrition  dans 
ses  Rapports  avec  I'lmmunity  "  (Masson  et  Cie.)  ;  discussion  on  Immunity, 
Brit.  Med.  Assoc.,  1904  ("  B.  M.  J.,"  September  10,  1904).  The  admirable 
abstracts  and  collected  articles  in  the  "  Central blatt  f.  Bakteriologie  " 
(Referate),  in  the  "  Bulletin  de  1'Institut  Pasteur,"  and  in  "  Folia  Haemato- 
logica,"  will  be  found  invaluable. 

Cold  and  Wet. — See  Trommsdorf,  Arch.  f.  Hyg.,  vol.  lix.,  p.  i,  and 
Vincent,  Bull.  Acad.  Med.,  1908.  Ciuca,  Comptes  Rendus  Soc.  Biol., 
vol.  Ixii.,  pp.  858,  883.  Alcohol. — See  Friedberger,  Congres  internat. 
d'Hyg.  and  Demog.  (Brux.),  1903.  Rubin,  Journ.  Inf.  Dis.,  1904,  p.  424. 
Trommsdorf  (vide  supra}.  Laitinen,  Zeit.  f.  Hyg.,  vol.  Iviii.,  1907,  p.  139. 

Anesthesia. — Snell,  Berlin.  Klin.  Woch.,  1903,  p.  212.  Rubin,  Journ. 
Inf.  Dis.,  vol.  i.,  p.  424. 

Ehrlich  (Trypanosomiasis,  Atreptic  Immunity,  etc.),  Harben  Lectures 
(H.  K.  Lewis). 

Walker,  Ainley,  Journ.  Hyg.,  vol.  iii.,  p.  52  ;  Cent.  f.  Bak.,  vol.  xxxiii., 
p.  297  ;  Journ.  Path.  Bact.,  1903,  p.  34. 

Papers  on  The  Early  Work  on  Immunity  against  Anthrax,  etc.,  will  be 
found  in  Microparasites  in  Disease,  New  Sydenham  Soc.,  1886.  See  also 
Pasteur,  Comptes  Rendus  de  1'Acad.  des  Sci.,  1880.  Pasteur,  Roux, 
and  Chamberland,  ibid.,  1883,  xcvii.  Rabies. — See  Sims  Woodhead's 
article  in  Clifford  Allbutt's  System  of  Medicine,  with  a  full  bibliography 
up  to  1906.  See  also  Chapter  XIV. 

A  useful  account  of  the  Use  of  Vaccines,  etc.,  in  Veterinary  Practice  is 
given  in  Jowett's  Blood-Serum  Therapy  (Bailliere,  Tindall  and  Cox,  1907). 

CHAPTER  II 

A  full  account  of  the  subject  will  be  found  in  Oppenheimer's  "  Toxine  und 
Antitoxine  "  (English  translation  by  Ainsworth  Mitchell  :  Charles  Griffin 
and  Co.).  This  contains  a  most  useful  bibliography  extending  to  1904. 

Antagonism  of  B.  pyocyaneus  and  B.  anthracis. — Woodhead  and  Wood, 
Edin.  Med.  Journ.,  1890.  Nasik  vibrio. — Kraus,  R.,  Centr.  f.  Bakt.  I.  O., 

421 


422  BIBLIOGRAPHY 

vol.  xxxiv.,  1903,  p.  488,  and  Rothberger,  ibid.,  vol.  xxxviii.,  1905,  p.  165. 
Absorption  of  Toxins  by  Tissue. — Ignowtowsky,  Cent.  f.  Bakt.  T.  O., 
vol.  xxxv.,  p.  4.  Vaillard,  quoted  by  Metchnikoff  (L'Immunite). 

Wassermanris  Experiment. — See  Chapter  IV. 

Combining  Reactions  of  Tetanolysin. — Ehrlich,  Berlin.  Klin.  Woch., 
1898,  p.  273.  Madsen,  Zeit.  f.  Hyg.,  1899,  xxxii.,  214. 

Action  of  Tetanus  Toxin  on  Frogs  at  Different  Temperatures. — Courmont 
and  Doyon,  Comptes  Rendus  de  la  Soc.  Biol.,  1893,  p.  618.  Morgenroth, 
Archives  Int.  de  Pharmacodyn,  1900,  p.  265.  Constitution  of  Toxin 
Molecule. — Ehrlich,  Croonian  Lecture,  Proc.  Roy.  Soc.,  1900.  Ibid., 
Congres  internat.  de  Med.,  Paris,  1900,  Klin.  Jahrbuch,  vol.  vi.  (Die  Werth- 
bemessung  des  Diphthericheilserums). 

Leucolysins  or  Leucotoxins. —  Neisser  and  Wechsberg,  Zeit.  f.  Hyg., 
vol.  xxxvi.,  1901,  p.  300.  Kerner,  Julius,  Cent.  f.  Bakt.  I.  O.,  vol.  xxxviii., 
p.  223.  Christian,  H.  A.,  Deut.  Arch.  f.  Klin.  Med.,  vol.  Ixxx.,  p.  333. 
Denys  and  van  de  Velde,  La  Cellule,  vol.  xi.,  p.  359. 

Bacterial  Hcemolysins  in  Relation  to  Toxicity. — Besredka,  Ann.  de  1'Inst. 
Past.,  vol.  xv.  Ruedinger,  Journ.  Amer.  Med.  .Assoc.,  1903.  Breton, 
Comptes  Rendus  de  la  Soc.  Biol.,  vol.  lv.,  p.  886.  Schlesinger,  Zeit.  f.  Hyg., 
vol.  xliv.,  p.  428.  Bacterial  H&molysins. — A  full  bibliography  will  be 
found  in  Oppenheimer,  and  a  good  general  account  of  the  subject  by 
Besredka,  Bull,  de  1'Inst.  Pasteur,  vol.  i.,  1903,  pp.  547,  579. 

Ricin. — Ehrlich,  Deut.  Med.  Woch.,  1891.  Fortschr.  d.  Med.,  1897. 
Stillmarck,  Arb.  pharm.  Inst.  Dorpat  (quoted  by  Oppenheimer).  Jacoby, 
Arch.  exp.  Path.,  xlvi.,  p.  28.  Osborne  and  Mandel,  Amer.  Journ.  Phys., 
vol.  x.,  p.  36. 

Serum  Toxin. — Cartwright  Wood.     See  Chapter  II. 

Endotoxins  (Pyocyaneus}. — Wassermann,  Zeit.  f.  Hyg.,  xxii.,  p.  263. 
Endotoxins  in  general:  Macfadyen,  Proc.  Roy.  Soc.,  1903,  p.  76;  1903, 
p.  351.  Macfadyen  and  Rowland,  Cent.  f.  Bakt.  I.  O.,  vol.  xxxiv.,  p.  618. 
Macfadyen,  Lancet,  1904,  p.  494.  Macfadyen  and  Rowland,  Journ.  Phys., 
vol.  xxiii.  Macfadyen,  B.  M.  J.,  1906,  p.  776.  Also  Vaughan  and  Wheeler, 
Journ.  Amer.  Med.  Assos.,  1905.  Ransom,  Deut.  Med.  Woch.,  1895,  P-  457- 
Petterson,  Cent.  f.  Bakt.,  vol.  xlvi.,  p.  405.  Pfeiffer  and  Friedberger,  Cent, 
f.  Bakt.  I.  O.,  vol.  xlvi.,  p.  98.  Metchnikoff,  Roux  and  Taurelli-Salembeni, 
Ann.  de  1'Inst.  Past.,  vol.  x.,  p.  257.  Besredka,  Ann.  Inst.  Past.,  1906, 
pp.  81,  304. 

See  also  the  discussion  on  Endotoxins  (Kraus  especially),  Cent.  f.  Bakt. 
(Ref.),  vol.  xlii. 

CHAPTER  III 

Methods  of  Preparing  Antitoxin,  etc. — Levaditi,  in  Kraus  and  Levaditi's 
Handbuch,  vol.  ii.,  p.  62.  Woodhead,  Report  of  M.  A.  B.,  1901.  Hewlett's 
Serumtherapy  (Churchill,  1903).  Dean,  Trans.  Path.  Soc.,  vol.  1L,  p.  15. 
Madsen,  Zeit.  f.  Hyg.,  vol.  xxiv.,  p.  425.  Hibbert,  Journ.  Exp.  Med\, 
vol.  vii.,  p.  176.  Martin,  Ann.  Inst.  Past.,  vol.  xii,,  p.  26.  Park  and 
Williams,  Journ.  Exp.  Med.,  1896,  No.  i.  Atkinson,  Journ.  Med.  Res., 
vol.  ix.,  p.  173.  Salomonsen  and  Madsen,  Ann.  Inst.  Past.,  vol.  xi., 
p.  315,  and  vol.  xii.,  p.  763.  Hibbert,  Journ.  Exp.  Med.,  vol.  vii.,  p.  176. 

Serum-toxin. — Cartwright  Wood,  Proc.  Roy.  Soc.,  vol.  lix.,  p.  290  ; 
Cent.  f.  Bakt.,  vol.  xxxi.,  p.  241. 

CHAPTER  IV 

Action  of  Ricin  on  Red  Blood-Corpuscles. — Ehrlich,  Fortsch.  der  Med., 
1897,  P-  41-  Of  Snake  Venom. — Stephens  and  Myers,  B.  M.  J.,  1898, 
vol.  Ixiii.,  p.  20.  Of  Eel  Serum. — Kossel,  Berlin.  Klin.  Woch.,  1898,  p.  152. 
Camus  and  Gley,  Ann.  Inst.  Past.,  1899,  P-  779-  Tchistovitch,  ibid.,  1899. 
Action  of  Leucocidin. — Neisser  and  Wechsberg,  Zeit.  f.  Hyg.,  1901,  p.  299. 


BIBLIOGRAPHY  423 

Filtration  Experiments. — Martin  and  Cherry,  B.  M.  J.,  1898,  p.  1120. 
Brodie,  Journ.  Path,  and  Bact.,  1897,  p.  460.  Action  of  Heat  on  Snake 
Venom,  etc. — Calmette,  Ann.  Inst.  Past.,  1895,  p.  225.  Martin  and 
Cherry,  Proc.  Roy.  Soc.,  1898.  Wassermann,  Zeit.  f.  Hyg.,  vol.  xxii., 
p.  263.  Marenghi.  Cent.  f.  Bakt.  I.  O.,  vol.  xxii.,  p.  521. 

Constitution  of  Diphtheria  Toxin. — Ehrlich,  Die  Wertbemessung  des 
Diphtherieheilserums,  and  numerous  other  papers,  some  of  the  more 
important  of  which  are  in  his  Collected  Papers.  Madsen,  B.  M.  J.,  1904, 
p.  567.  Oppenheim,  Toxin  and  Antitoxin.  Gruber  and  Pirquet,  Munch, 
med.  Woch.,  vol.  1.,  pp.  1193,  1259. 

Arrhenius  and  Madsen' s  Theories  are  discussed  at  great  length  in  the 
former's  Immuno-Chemistry  (Macmillan),  where  full  references  are  given. 
See  also  Madsen,  B.  M.  J.,  1904,  September  10.  Myers,  Lancet,  1898,  vol.  ii., 
p.  23,  and  Journ.  of  Path,  and  Bact.,  1900.  Mouton,  Bull,  de  1'Inst.  Past., 
vol.  v.,  1907,  p.  449  (with  bibliography).  Arrhenius  and  Madsen,  Zeit. 
f.  Phys.  Chem.,  vol.  xliv.,  p.  6.  Gruber  and  v.  Pirquet,  Miinch.  Med. 
Woch.,  vol.  1.,  p.  1193.  Arrhenius,  Bull.  Inst.  Past.,  vol.  ii.,  1904,  p.  553 
(good  general  account).  Madsen  and  Walbum,  Cent.  f.  Bakt.  I.  O.(  vol. 
xxxvi.,  p.  242.  Mainwaring,  W.  H.,  Journ.  Inf.  Dis.,  vol.  iii.,  p.  638. 
Morgenroth  and  Pane,  Biochem.  Zeit.,  vol.  i.,  p.  354.  Nernst,  Zeit.  f. 
Electrochemie,  x.,  p.  177.  Ehrlich's  Reply  to  Arrhenius  Theory,  Berlin. 
Klin.  Woch.,  1903,  Nos.  35  and  37  (XXXVII.  in  Collected  Studies). 

Bordet's  Views. — Bordet,  Ann.  Inst.  Past.,  vol.  xvii.,  p.  161.  Eisenberg, 
Cent.  f.  Bakt.,  xxxiv.,  p.  259.  Biltz,  Zeit.  f.  Phys.  Chem.,  1904,  p.  615. 
See  also  Chapter  XII. 


CHAPTER  V 

Action  of  Electricity  on  Toxins. — Kruger,  Deut.  Med.  Woch.,  1895,  p.  331. 
D'Arsonval  and  Charrin,  Comptes  Rendus  de  la  Soc.  de  Biol.,  1896,  pp.  122, 
280.  Marmier,  Ann.  de  1'Inst.  Past.,  vol.  x.,  p.  468.  Knorr,  Miinch. 
Med.  Woch.,  1898,  p.  321. 

Antibodies  in  Normal  Blood. — Metchnikoff,  L'lmmunite,  p.  598.  Neisser, 
Deut.  Med.  Woch.,  1900,  p.  791.  Cobbett,  Lancet,  1899,  p.  332.  Ibid., 
Cent.  f.  Bakt.,  vol.  xxvi.,  p.  548.  Meade,  Bolton,  Journ.  Exp.  Med., 
vol.  i.,  p.  543. 

Regeneration  of  Antitoxin  after  Bleedings. — Roux  and  Vaillard,  Ann.  Inst. 
Past.,  vol.  vii.,  p.  64.  Salom onsen  and  Madsen,  ibid.,  vol.  xii.,  p.  763. 
Action  of  Pilocarpin. — Salomonsen  and  Madsen,  Comptes  Rendus  de 
1'Acad.  des  Sciences,  vol.  cxxv.,  p.  122. 

Side-Chain  Theory. — Ehrlich,  Croonian  Lecture,  Proc.  Roy.  Soc.,  vol. 
Ixvi.,  p.  424.;  Ver.  f.  Innere  Med.  Berlin,  1901.  Numerous  articles  in 
Collected  Studies.  See  also  Aschoffs  Ehrlich's  Seitenkettentheorie 
(Fischer,  1902),  with  a  very  full  bibliography.  Levaditi,  La  Nutrition  dans 
ses  rapports  avec  I'lmmunite  (Masson  and  Cie.),  which  also  gives  numerous 
references.  Wassermann,  Berlin.  Klin.  Woch.,  1898.  Plimmer,  Journ. 
Path,  and  Bact.,  1898,  p.  489.  Figs.  22,  23,  24  are  from  Emery,  The 
Specific  Antibodies,  St.  Bart.'s  Hosp.  Journ.,  1902.  Weigert,  Verhandlung 
der  ges.  Deutscher  Naturforscher  und  Aerzte,  1896.  Bruck,  Zeit.  f.  Hyg., 
vol.  xlvi.,  p.  176. 

Union  of  Tetanus  Toxin  with  Brain  Substance. — Wassermann  and 
Takaki,  Berlin.  Klin.  Woch.,  1898.  Metchnikoff,  Ann.  Inst.  Past.,  vol.  xii., 
pp.  81,  263.  Marie,  ibid.,  p.  91.  Courmont  and  Doyon,  Comptes  Rendus 
de  la  Soc.  Biol.,  1898,  p.  602.  Joukowsky,  Ann.  Inst.  Past.,  vol.  xiii., 
p.  464.  Morax  and  Marie,  ibid.,  vol.  xvii.,  p.  335,  and  Comptes  Rendus 
Soc.  Biol.,  vol.  liv.,  p.  1535.  Dmitrevsky,  Ann,  Inst.  Past.,  vol.  xvii., 
p.  148.  Besredka,  ibid,  p.  138.  Muller,  Cent.  f.  Bakt.  I.  O.,  vol.  xxxiv., 
p.  567.  Landsteiner  and  Boteri,  ibid.,  vol.  xlii.,  p.  562.  Wolff-Eisner  and 
Rosenbaum,  Berlin.  Klin.  Woch.,  1906,  p.  945.  Takaki,  Beitr.  z.  Chem. 


424  BIBLIOGRAPHY 

Phys.  und  Path.,  vol.  xi.,  p.  238.  Morax  and  Tiffaneau,  Comptes  Rendus 
de  la  Soc.  Biol.,  vol.  Ixii.,  p.  15.  Noon,  Journ.  Hyg.,  vol.  vii.,  p.  101. 

Romer's  Experiments. — Arch.  f.  Opthal.,  vol.  lii.,  p.  72. 

Antispermotoxin. — Metchnikoff,  L'Immunite,  p.  130.  Blum,  Beit.  z. 
Chem.  Phys.,  1904.  Vaillard  and  Vincent,  Ann.  Inst.  Past.,  vol.  v.,  p.  i. 
Besredka,  Ann.  Inst.  Past.,  vol.  xiii.,  pp.  49,  209. 


CHAPTER  VI 

Non-Specific  Processes. — Herter,  Lectures  on  Chemical  Pathology 
(Smith,  Elder,  1902). 

Function  of  Liver. — Brunton,  Sir  L.,  and  Bokenham,  Journ.  Path.  Bact., 
1905,  p.  50. 

Antitoxin  in  Blood  after  Diphtheria,  etc. — Wassermann,  Zeit.  f.  Hyg., 
vol.  xix.,  p.  408.  Abel,  Deut.  Med.  Woch.,  1894,  pp.  899,  936.  Vincenzi, 
ibid.,  1898,  p.  247.  Knorr,  Munch.  Med.  Woch.,  1898,  p.  363. 

Absence  of  Correlation  between  Immunity  and  Antitoxin. — Roux  and 
Vaillard,  Ann.  Inst.  Past.,  vol.  vii.,  p.  64.  Behring  and  Kitashima,  Berlin. 
Klin.  Woch,  1901,  p.  157.  Metchnikoff,  L'Immunite,  pp.  386  et  seq. 
Behring,  Allgemeine  Therapie  der  Infectionskrankheiten,  in  Eulenberg 
and  Samuel's  Lehrbuch  der  Allg.  Therapie. 

Leucocytes  in  Intoxications. — Metchnikoff,  loc.  cit.,  p.  413,  where  nu- 
merous references  are  given.  Besredka,  Ann.  Inst.  Past.,  vol.  xiii.,  pp.  49, 
205  and  465.  Dean,  Journ.  Path.  Bact.,  1908,  p.  154.  Ewing,  Clinical 
Pathology  of  Blood  (Kimpton,  1904),  p.  292.  Vincent,  Ann.  Inst.  Past, 
vol.  xviii.,  p.  450. 

Stimulins. — Metchnikoff,  loc.  cit.,  p.  284. 

Wassermann,  N.  Y.  Med.  Journ.,  1904. 

Immunization  to  Eel  Serum. — Tchistovitch,  Ann.  Inst.  Past.,  vol.  xiii., 
p.  406. 

Specific  Processes. — See  Ehrlich's  Croonian  Lecture,  Harben  Lectures, 
and  various  papers  in  his  Collected  Studies.  Wassermann  and  Bruck, 
Deut.  Med.  Woch.,  1904,  p.  764.  Jacoby,  Beit.  z.  Chem.  Phys.,  vol.  vi., 
p.  113.  Bruck,  Zeit.  f.  Hyg.,  vol.  xlix,  p.  282.  Ricketts,  Trans.  Chicago 
Path.  Soc.,  vol.  vi.  Metchnikoff,  L'Immunite,  Chapters  XI,  XII.  Leva- 
diti's  L'Immunite  dans  ses  Rapports  avec  la  Nutrition. 

Passive  Immunity. — McClintock  and   King,   Journ.   Inf.   Dis.,  vol.   iii. 
p.  701.     Goodman,  ibid.,  vol.  v.,  p.   184.     Bulloch,  Journ.  Path.  Bact., 
1898,  p.  274.     Schiitze,   Koch's  Festschrift,  p.  657.     Pfeiffer  and  Fried  - 
berger,  Cent.  f.  Bakt.  I.  O.,  vol.  xxxvii.,  p.  131.     Wassermann  and  Bruck, 
Zeit.  f.  Hyg.,  vol.  1.,  p.  309.     Weil-Halle  and  Lemaire,  Comptes  Rendus 
Soc.  Biol.,  1906,  p.  114.     Henderson  Smith,  Journ.  Hyg.,  vol.  vii.,  p.  205 
Goodman,  Journ.  Inf.  Dis.,  vol.  v.,  p.  184. 

Susceptibility  to  Tetanus  Toxins. — Knorr,  Munch.  Med.  Woch.,  1898, 
pp.  321,  362.  Behring,  Fortschr.  der  Med.,  vol.  xvii.,  p.  501.  Behring's 
Beitrage,  August,  1903.  See  also  Chapters  V.,  XIV. 


CHAPTER  VII 

Alexins.— Nuttall,  Zeit.  f.  Hyg.,  vol.  iv.,  1888,  p.  353.  Behring,  Cent, 
f.  Klin.  Med.,  1888,  No.  32.  Behring  and  Nissen,  Zeit.  f.  Hyg., 
vol.  viii.,  p.  412.  Buchner,  Cent.  f.  Bakt.  I.  O.,  vol.  v.,  p.  817.  Ibid., 
Arch.  f.  Hyg.,  vol.  x.,  p.  84.  Ibid.,  Arch.  f.  Hyg.,  vol.  xvii.,  p.  112.  Ibid., 
Munch.  Med.  Woch.,  vol.  xlvii.,  p.  277.  Lubarsch,  Cent.  f.  Bakt.,  vol.  vi., 
p.  481,  529.  Pfeiffer,  Zeit.  f.  Hyg.,  vol.  xviii.  ;  Deut.  Med.  Woch.,  1896, 
pp.  97,  119.  Bordet,  Ann.  Inst.  Past.,  vol.  ix.,  p.  462,  and  ibid.,  vol.  xii., 
p.  688.  Landsteiner,  Cent.  f.  Bakt.  I.  O.,  vol.  xxv.,  p.  546. 

Ehrlich's  Researches  are  given  in  his  Collected  Studies,  the  main  chapters 


BIBLIOGRAPHY  425 

being  I. -VIII.,  XVII.,  XIX.,  XXI.,  XXII.,  XXXII.,  XXXIII.,  and  XL. 

Marino,  Ann.  Inst.  Past.,  vol.  xvii.,  p.  321. 

A  full  account  of  the  subject  of  Hcemolysins,  with  an  excellent  biblio- 
graphy, is  given  by  Sachs  in  Kraus  and  Levaditi,  vol.  i.(  and  the  work  of 
Muir  and  his  school  has  just  been  published  in  collected  form  (The  Oxford 
Press,  1909).  See  also  Flexner  and  Noguchi,  Journ.  Exp.  Med.,  vol.  vi. 
Kyes,  Berlin.  Klin.  Woch.,  1902  (reprinted  in  Ehrlich's  Studies).  Kyes 
and  Sachs,  Berlin.  Klin.  Woch.,  1903,  p.  21,  57,  82.  Kyes,  Berlin.  Klin. 
Woch,  1903,  p.  956,  982.  Bordet's  Views. — Bordet,  Ann.  Inst.  Past., 
vol.  xiii.,  1899,  PP-  225>  273  >  v°l-  xiv.,  p.  257  ;  1906,  p.  467.  Muir  and 
Browning,  Proc.  Roy.  Soc.,  vol.  Ixxiv.,  p.  298  ;  Journ.  Path,  and  Bact.^. 
vol.  xiii.,  p.  76.  Muir,  Lancet,  vol.  clxv.,  p.  446,  and  B.  M.  J.,  Septem- 
ber 10,  1904.  Muir  and  Ferguson,  Journ.  Path,  and  Bact.,  1906,  p.  84. 
Metchnikoff,  L'Immunite,  Chapters  VII.,  VIII. 

Bordet  and  Gengou' s  Phenomenon  (Fixation  of  Complement). — Bordet, 
Ann.  Inst.  Past.,  vol.  xv.,  p.  289.  Bordet  and  Gengou,  C.  R.  Acad.  Sci., 
vol.  cxxxvii.,  p.  351.  Gengou,  Berlin.  Klin.  Woch.,  1906,  p.  1532.  Muir 
and  Martin,  Journ.  of  Hyg.,  vol.  vi.,  p.  265.  Heller  and  Tomarkin,  Deut. 
Med.  Woch.,  1907,  p.  795.  Cruveilhier,  Comptes  Rendus  Soc.  Bio., 
vol.  Ixii.,  p.  1027.  Schutze,  Berlin.  Klin.  Woch.,  1907,  p.  800.  Seligmann, 
ibid.,  1907,  p.  1013.  Widal  and  le  Sourd,  Comptes  Rendus  de  la  Soc. 
Biol.,  1901,  pp.  673,  841.  Wassermann  and  Bruck,  Deut.  Med.  Woch., 
1906,  p.  449.  Bruck,  Deut.  Med.  Woch.,  1906,  June,  p.  945.  Also  Camus 
and  Pagnier,  Comptes  Rendus  Soc.  Biol.,  1901,  July. 

For  References  re  Gengou' s  Phenomena  see  also  Chapter  IX. 

Deviation  by  Toxins,  etc. — Armand-Delille,  Comptes  Rendus  Soc.  Biol., 
1908.  Poyerski,  ibid.,  1908,  p.  896.  Weinberg  and  Parvu,  ibid.,  Novem- 
ber, 1908.  Laubry  and  Parvu,  Soc.  Med.  des  Hop.,  December,  1908. 

In  Explanation  of  Complementoid. — Moreschi,  Berlin.  Klin.  Woch., 
vol.  xiii.,  September,  1905,  p.  1181.  Gay,  Cent.  f.  Bakt.,  vol.  xxxix.,  1905, 
pp.  172,  603  ;  also  vol.  xl.,  p.  695.  Pfeiffer  and  Friedberger,  Deut. 
Med.  Woch.,  1905,  p.  6.  Besredka,  Ann.  Inst.  Past.,  1905.  Sachs,  Deut. 
Med.  Woch.,  May,  1908.  Pfeiffer  and  Friedberger,  Deut.  Med.  Woch., 
1905,  p.  1145.  Bordet,  Ann.  Inst.  Past.,  vol.  xv.,  p.  289.  Sachs,  Cent.  f. 
Bakt.  I.  O.,  vol.  xl.,  p.  125.  Bordet,  Berlin.  Klin.  Woch.,  1906,  p.  17. 
Pfeiffer  and  Moreschi,  Berlin.  Klin.  Woch.,  1906,  p.  33. 

Deviation  of  Complement. — Loffler  and  Abel,  Cent.  f.  Bakt.  I.  O.(  vol.  xix., 
p.  51.  Pfeiffer,  Zeit.  f.  Hyg.,  vol.  xx.,  p.  198.  Neisser  and  Wechsberg, 
Munch.  Med.  Woch.,  1901.  Lipstein,  Cent.  f.  Bakt.  I.  O.,  vol.  xxxi.,  p.  460. 
Morgenroth,  ibid.,  vol.  xxxv.,  p.  501.  Myers  and  Stephens,  Journ.  Path. 
Bact.,  vol.  v.  Kyes,  Berlin.  Klin.  Woch.,  1902,  and  Kyes  and  Sachs,  ibid., 

1903  (both   these   are   in  Ehrlich's   Collected   Studies).     Meakins,   Johns 
Hopkins  Bull.,  1907,  p.  259. 

Origin  of  Complement,  Alexin,  etc. —  Hankin,  Cent.  f.  Bakt.  I.  O.,  xii. 
and  xiv.,  p.  853.  Denys  and  Havet,  La  Cellule,  1894,  vol.  x.,  p.  7. 
Havet,  ibid.,  1894,  vol.  x.  Denys,  Cent.  f.  Bakt.  I.  O.,  vol.  xvi.,  p.  781. 
Buchner,  Munch.  Med.  Woch.,  1894,  p.  589;  Metchnikoff,  L'lmmunite. 
Bulloch,  Trans.  Path.  Soc.,  1901,  p.  208;  ibid.,  B.  M.  J.,  September  10, 

1904  (with    full    bibliography).     Longcope,    Journ.    of    Hyg.,    vol.    iii., 
p.    28.     Guseff,    quoted    by  "  Petrie,   loc.   cit.     Briscoe,    Orth   Festschrift, 
1903.    Levaditi,  Ann.  Inst.  Past.,  xvii.,  p.  187.     Korschun  and  Morgenroth, 
Berlin.  Klin.  Woch.,  1902.     Marino,  Comptes  Rendus  Soc.  Biol.,  vol.  lv., 
p.   689.     Schattenfroh,   Arch.   f.   Hyg.,    vol.    xxxi.,   p.    i  ;    ibid.,    xxvii., 
p.  234;    ibid.,   Munch.   Med.  Woch.,    1897,   P-  4T4  '>  ibid.,   1898,  p.  1109  ; 
ibid.,    1897,    P-    4'    ibid.,    Arch.    f.    Hyg.,    vol.    xxxv.,    p.    135,    Petrie, 
Journ.    Path.     Bact.,    vol.     ix.,    p.     130.       Lastschenko,    Munch.    Med. 
Woch.,  1899,  p.  475  ;  ibid.,  Arch.  f.  Hyg.,  xxxvii.,  p.  290.     Lambotte,  Cent, 
f.  Bakt.  I.  O.,  vol.  xxxiv.,  p.  453.     Lambotte  and  Stienon,  Cent.  f.  Bakt. 
I.  O.,  vol.  xl.,  p.  224.     Donath  and  Landsteiner,  Zeit.  f.  Hyg.,  vol.  xliii., 
p.    552.     Lowit  and   Schwarz,   Zeit.   f.   Heilk.,   vol.   xxiv.,   pp.   205,    301. 


426  BIBLIOGRAPHY 

Gengou,  Ann.  Inst.  Past.,  vol.  xv.,  p.  68  ;  ibid.,  vol.  xv.,  p.  232.  Falloise, 
Comptes  Rendus  Soc.  Biol.,  vol.  Ivi.,  p.  324.  Falloise,  Bull,  de  1'Acad. 
Royale  de  Belgique,  1903.  Levaditi,  Ann.  Inst.  Past.,  1901,  vol.  xv., 
p.  894,  and  vol.  xvi.,  p.  233,  1902.  Ainley  Walker,  Journ.  Hyg., 
vol.  iii.,  p.  52,  and  Cent.  f.  Bakt.,  vol.  xxxiii.,  p.  297.  See  also  Hahn, 
Arch.  f.  Hyg.,  vol.  xxv.,  p.  105.  Wauters,  Arch,  de  Med.  Exp.,  vol.  x., 
p.  751.  Moxter,  Deut.  Med.  Woch.,  1899,  p.  687.  Tromsdorff, 
Arch.  f.  Hyg.,  vol.  xl.,  p.  382.  Van  de  Velde,  Cent.  f.  Bakt.  I.  O., 
vol.  xxiii.,  p.  692.  Bail,  Hyg.  Rundschau,  vol.  viii.,  p.  1066.  Sweet, 
Cent.  f.  Bakt.  I.  O.,  vol.  xxxiii.,  p.  208.  Malvoz,  Ann.  Inst.  Past.,  vol.  xvi., 
p.  623.  Lazar,  Wien.  Klin.  Woch.,  1904,  p.  439.  Kanthack,  vide 
Chapter  X.  Steinhardt,  Journ.  Med.  Research,  vol.  xix.,  p.  161. 
Gousseff,  abstract  in  Bull.  Inst.  Past.,  vol.  i.,  p.  175.  Longcope,  Journ. 
Hyg.,  vol.  iii.,  p.  28. 

Origin  of  Immune  Bodies,  etc. — Pfeiffer    and  Marx,  Deut.  Med.  Woch., 

1898,  p.  47  ;  Deutsch.  Ann.  Inst.  Past.,  vol.  xiii.,  p.  689  ;  and  Cent.  f. 
Bakt.  I.  O.,  vol.  xxviii.,  p.  45.     Kraus  and  Schiffmann,  Ann.  Inst.  Past., 
vol.  xx.,  p.  226.     Bulloch,  vide  ante.     Wassermann,  Deut.  Med.  Woch., 

1899,  p.    141.     Wassermann   and  Citron,   Zeit.   f.   Hyg.,  vol.  1.,   p.    331. 
Pfeiffer  and  Marx,  Zeit.  f.  Hyg.,  vol.  xxvii.,  p.  272.     Donath  and  Land 
steiner,  Zeit.  f.  Hyg.,  vol.  xliii.,  p.  552.     Kraus  and  Schiffmann,  Ann.  Inst. 
Past.,  vol.  xx.,  p.  226.     Kraus  and  Levaditi,  Comptes  Rendus  Acad.  Sci., 
vol.  cxxxviii.     Emden,  Zeit.  f.  Hyg.,  vol.  xxx.,  p.  19. 

Methods  (Haemolysis}. — Papers  in  Ehrlich's  Collected  Studies,  especially 
Chapter  XXIX.  (Morgenroth) .  Sachs  in  Kraus  and  Levaditi's  Handbuch 
der  Technik  and  Methodik  der  Immunitatsforschung  (Fischer,  Jena, 
1907).  Moro,  Munch.  Med.  Woch.,  vol.  liv.,  p.  1026.  Gay  and  Ayer, 
Journ.  Med.  Research,  vol.  xvii.,  p.  341.  Longcope,  Univ.  of  Penn.  Med. 
Bull.,  xv.,  p.  331. 

Methods  (Bacteriolysis}. — Ehrlich's  Studies,  Chapters  IX.,  XXX.  (Neisser 
and  Wechsberg).  Klien,  Johns  Hopkins  Bull.,  1907,  p.  245.  Stern  and 
Korte,  Berlin.  Klin.  Woch.,  1904,  p.  213.  Wright,  Lancet,  December, 
1900  ;  ibid.,  1901,  March  2  and  September  14  ;  ibid.,  Proc.  Roy.  Soc., 
Ixxi.,  p.  54.  Gay  and  Ayer,  loc.  cit.  Andrewes  and  Gordon,  Report  of 
L.G.B.  (supplement),  1906-7,  p.  141.  Goodwin,  Proc.  N.  Y.  Path.  Soc.. 
vol.  v. 

Cytolysins,  etc.  ;  Leucolysins. — Metchnikoff,  L'lmmunite.  Funk,  Cent, 
f.  Bakt.  I.  O.,  vol.  xxvii.,  p.  670.  Flexner,  Univ.  of  Penns.  Med.  Bull., 
vol.  xv.  Bunting,  ibid.,  vol.  xvi.,  p.  200.  Goodman,  Journ.  Inf.  Dis., 
vol.  v.,  p.  173.  Christian,  Deut.  Arch.  f.  Klin.  Med.,  vol.  Ixxx.,  p.  333. 

Spermotoxin. —  Metchnikoff,  Ann.  Inst.  Past.,  vol.  xiv.,  p.  i,  369. 
Metalnikoff,  ibid.,  p.  577.  Moxter,  Deut.  Med.  Woch,  1900,  p.  61. 
Landsteiner,  Cent.  f.  Bakt.  I.  O.,  vol.  xxv.,  p.  546.  London,  Arch,  de 
Sci.  Biol.  St.  Petersburg,  vol.  ix.  Weichardt,  Ann.  Inst.  Past.,  vol.  xv., 
p.  8^3. 

3  ^'specificity  and  General. — Sachs,  Biochem,  Cent.,  1903.  Pearce,  Journ. 
/.^  Exp.  Med.,  vol.  viii.  ;  ibid.,  Journ.  Med.  Res.,  vol.  xii.,  pp.  i,  329.  Beebe, 
Journ.  Exp.  Med.,  vol.  vii.,  p.  730.  Armand-Delille  and  Leenhardt,  C.  R. 
Soc.  Biol.,  vol.  Ixii.,  p.  31.  Woltmann,  Journ.  Exp.  Med.,  vol.  vii.,  p.  119. 
Forsner,  Munch.  Med.'  Woch.,  vol.  Hi.,  p.  892.  Flexner  and  Noguchi, 
Journ.  Med.  Res.,  vol.  ix.,  p.  257.  Bierry  and  Pettit,  C.  R.  Soc.  Biol. 
vol.  Ivi.,  p.  238.  Dudgeon,  Panton,  and  Ross,  Proc.  Roy.  Soc.  Med., 
vol.  ii.,  No.  2. 

Trichotoxin. — Von  Dungern,  Munch.  Med.  Woch.,  1899.  Hoyton, 
B.  M.  J.,  1902. 

Nephrotoxin. — Nefedieff,  Ann.  Inst.  Past.,  vol.  xv.,  p.  17.  Ascoli  and 
Figari,  Berlin.  Klin.  Woch.,  1902.  Lindemann,  Cent.  f.  Allg.  Path., 
vol.  vi.,  p.  184.  Pearce,  Univ.  Penns.  Med.  Bull.,  vol.  xvi.,  p.  217. 
Bierry,  C.  R.  Acad.  Sci.,  vol.  cxxxii.  Bierry,  C.  R.  Soc.  Biol.,  vol. 
lv.,  p.  496.  Le  Play  and  Corpechot,  ibid.,  p.  206.  Sheldon,  Amos, 


BIBLIOGRAPHY  427 

Reports  of  Med.  Staff,  Egyptian  San.  Council,  1906.  Albarran  and 
Bernard,  Arch,  de  Med.  Exp.,  vol.  xv.,  p.  13.  Woltmann,  Journ.  Exp. 
Med.,  vol.  vii.,  p.  119. 

Gastrotoxin. — Bolton,  Proc.  Roy.  Soc.,  vol.  Ixxvii.,  p.  426,  and  Ixxix., 
p.  533  ;  ibid.,  Proc.  Roy.  Soc.  Med.,  vol.  ii.,  No.  2.  Theobary  and  Bates, 
Comptes  Rendus  Soc.  Biol.,  1903,  p.  459. 

Anti-intestinal  Serum. — Belonowski,  Comptes  Rendus  Soc.  Biol.,  1907, 
P-  9- 

Syncytiolysin. — Liepmann,  Deut.  Med.  Woch.,  1902,  p.  911.  Weichardt, 
ibid.,  1902,  p.  624.  Ascoli,  Cent.  f.  Gynekol.,  1902.  Wormser,  Munch. 
Med.  Woch.,  1904,  p.  7. 

Neurotoxin. — Delezenne,  Ann.  Inst.  Past.,  vol.  xiv.,  p.  686  ;  ibid.,  Comptes 
Rendus  Soc.  Biol.,  1901,  p.  1161.  Armand-Delille,  Ann.  Inst.  Past., 
vol.  xx.,  p.  838  ;  ibid.,  Enriquer  and  Sicard,  Comptes  Rendus  Soc.  Biol., 

1900.  Pirone,  Arch.  Sci.  Biol.,  vol.  x.,  p.  75. 

For  Peripheral  Nerves. — Schmidt,  Ann.  Inst.  Past.,  vol.  xx.,  p.  601. 

Ophthalmotoxin. — Bram  Pusey,  quoted  by  Ricketts.  Le  Play  and 
Corpechot,  Comptes  Rendus  Soc.  Biol.,  1904,  p.  1021.  Golovine,  Russie 
Vratch,  1904,  abstracted  in  Bull.  Inst.  Pasteur,  vol.  ii.,  p.  1009. 

Hepatotoxin. — Delezenne,  Comptes  Rendus  Acad.  Sciences,  vol.  cxxxi., 
p.  427.  Pease  and  Pearce,  Journ.  Inf.  Dis.,  vol.  iii.,  p.  619.  Bolton, 
Proc.  Roy.  Soc.,  vol.  Ixxiv.,  p.  135.  Bierry  and  Mayer,  Comptes  Rendus 
Soc.  Biol.,  vol.  Ivi.,  p.  1016. 

Adrenotoxic  Serum,. — Bigart  and  Bernard,  Comptes  Rendus  Soc.  Biol., 

1901,  p.  161.     Yates,  Univ.  Penns.  Med.  Bull.,  vol.  xvi.,  p.  195. 
Thyrotoxic  Serum. — Gontscharnkow,  Cent.  f.  Allg.  Path.,  vol.  lix.,  p.  76. 

Portis,  Journ.  Inf.  Dis.,  vol.  i.,  p.  127. 

CHAPTER  VIII 

Gruber  and  Durham,  Munch.  Med.  Woch.,  1896,  p.  285  ;  ibid.,  1899, 
p.  1829.  Charrin  and  Roger,  Comptes  Rendus  Soc.  Biol.,  1889,  p.  667. 
Metchnikoff,  Ann.  Inst.  Past.,  vol.  v.,  p.  473.  Durham,  Journ.  Path. 
Bact.,  vol.  iv.,  p.  13,  and  vol.  vii.,  p.  240.  Grunbaum,  Lancet,  Septem- 
ber 19,  1896;  ibid.,  Munch.  Med.  Woch.,  1897,  No-  T3- 

Group  Reactions. — Pfaundler,  Munch.  Med.  Woch.,  1899,  November  15, 
p.  472.  Posselt  and  Sagasser,  Wien.  Klin.  Woch.,  1903,  p.  691.  Park, 
Journ.  Inf.  Dis.,  1906,  February,  p.  i.  Frouin,  Comptes  Rendus  Soc. 
Biol.,  vol.  Ixii.,  p.  154.  Crendiropoulo  and  Amos,  Reports  of  Egyptian 
Sanitary  Council,  1906.  Bordet,  Ann.  Inst.  Past.,  vol.  xiii.,  p.  225. 

Bacterio-precipitins. — Kraus,  Wien.  Klin.  Woch.,  1897,  August  12. 
Norris,  Journ.  Inf.  Dis.,  vol.  i.,  p.  463.  See  Chapter  IX. 

Agglutination  of  Flagella. — Smith  and  Reagh,  Journ.  Med.  Res.,  vol.  x., 
p.  89.  Buxton  and  Torrey,  Journ.  Med.  Res.,  vol.  xiv. 

Theories  as  to  the  Mechanism  of  the  Process. — Nicolle,  Ann.  Inst.  Past., 
vol.  xii.,  p.  161.  Paltauf,  Wien.  Klin.  Woch.,  1897.  Dineur,  Bull.  Acad. 
Med.  Belg.,  1898,  p.  652.  Bordet,  Ann.  Inst.  Past.,  vol.  x.,  p.  195,  and 
vol.  xiii.,  p.  225  (the  latter  especially).  Lowit,  Cent.  f.  Bakt.  I.  O.,  vol. 
xxxiv.,  pp.  156,  251.  Kraus  and  Joachim,  ibid.,  vol.  xxxvi.,  p.  662,  and 
xxxvii.,  p.  71. 

Site  of  Origin  of  Agglutinin. — Pfeiffer  and  Marx,  Deut.  Med.  Woch., 
1898,  p.  47.  Emden,  Zeit.  f.  Hyg.,  vol.  xxx.  Wassermann,  Deut.  Med. 
Woch.,  1899,  p.  141.  Deutsch,  Cent.  f.  Bakt.,  vol.  xxviii.,  p,  45.  Ruffer 
and  Crendiropoulo,  vide  ante. 

Colloid  Chemistry. — Biltz,  Zeit.  f.  Phys.  Chem.,  vol.  xlviii.,  p.  615. 
Neisser  and  Friedemann,  Munch.  Med.  Woch.,  1904,  p.  827.  Bechhokl, 
Zeit.  f.  Phys.  Chem.,  vol.  xlviii.,  p.  385.  See  also  Chapter  XII. 

Absorption  Test. — Castellani,  Zeit.  f.  Hyg.,  vol.  xl.,  p.  i. 

Park,  Journ.  Med.  Res.,  vol.  vii.     Hirschbruch,  Arch.  f.  Hyg.,  vol.  Ivi., 


428  BIBLIOGRAPHY 

p.  280.  Ballner,  Arch.  f.  Hyg.,  vol.  li.,  p.  245.  Lowit,  Cent.  f.  Bakt.  I.  O. 
vol.  xxxiv.,  pp.  156,  251. 

Constitution  of  Agglutinins,  A  gglutinoids ,  etc. — Wassermann,  Zeit.  f. 
Hyg.,  vol.  xlii.,  p.  267.  Buxton  and  Vaughan,  Journ.  Med.  Res.,  vol.  xii., 
p.  115.  Eisenberg  and  Volk,  Zeit.  f.  Hyg.,  vol.  xl.  Shibayama,  Cent.  f. 
Bakt.  I.  O.,  vol.  xlii.,  pp.  68,  144.  Joos,  Cent.  f.  Bakt.  I.  O.,  vol.  xxxiii., 
p.  762  ;  ibid.,  Zeit.  f.  Hyg.,  vol.  xxxvi.,  p.  422.  Scheller,  Cent.  f.  Bakt.  I.  O., 
vol.  xxxvi.,  p.  694.  Smith  and  Reagh,  Journ.  Med.  Res.,  vol.  x.,  p.  89. 
Buxton  and  Torrey,  Journ.  Med.  Res.,  vol.  xiv.,  April.  Dreyer  and 
Jex-Blake,  vide  Dreyer,  B.  M.  J.,  September  10,  1904,  p.  564  ;  Journ. 
Path.  Bact.,  vol.  xi.,  p.  i. 

Modifications  of  Bacteria  grown  in  Agglutinating  Serum. — Ainley  Walker, 
Journ.  Path.  Bact.,  vol.  viii.,  p.  34.  Welch,  Johns  Hopkins  Bull.,  vol. 
xiii.,  p.  291.  Muller,  Munch.  Med.  Woch.,  1903,  p.  56.  Bail,  Arch.  f. 
Hyg.,  vol.  xlii.,  p.  307.  Landsteiner,  Wien.  Klin.  Woch.,  1897,  P-  439- 
Marshall  and  Knox,  Journ.  Med.  Res.,  vol.  xv.,  p.  325.  See  also 
Chapter  XIII. 

Htzmagglutinins. — Landsteiner,  Cent.  f.  Bakt.  I.  O.,  vol.  xxvii.,  p.  357. 
Landsteiner  and  Leiner,  ibid.,  vol.  xxxviii.,  p.  548.  Hektoen,  Journ.'  Inf. 
Dis.,  vol.  iv.,  p.  297.  Gay,  Journ.  Med.  Res.,  vol.  xvii.,  p.  321.  Peskind, 
Amer.  Journ.  Phys.,  1903.  Biffi,  Ann.  d'Ig.  Sperim.,  vol.  xiii.,  abstracted 
in  Bull.  Inst.  Past.,  vol.  i.,  p.  526.  Shattock,  Journ.  Path.  Bact.,  vol.  vi., 
p.  303.  Ford  and  Halsey,  Journ.  Med.  Res.,  vol.  xi.,  p.  403.  Eisenberg, 
Wien.  Klin.  Woch.,  1901,  p.  1020.  Griinbaum,  B.  M.  J.,  1900,  p.  1089. 


CHAPTER  IX 

Precipitins  in  Normal  Sera. — Hoke,  Wien.  Klin.  Woch.,  vol.  xx.,  p.  347  ; 
Rodet,  Comptes  Rendus  de  la  Soc.  Biol.,  vol.  Iv.,  p.  1626.  Noguchi,  Bull. 
Univ.  Penns.,  vol.  xv.,  p.  301.  Ascoli,  abstracted  in  Bull.  Inst.  Past., 
vol.  i.,  p.  343. 

Specificity  of  Serum  Precipitins. —  Vide  Nuttall,  loc.  cit.,  in  which  the 
main  references  are  given.  Uhlenhuth,  Deut.  Med.  Woch.,  1901,  pp.- 82, 
499.  Wassermann  and  Schiitze,  Berlin.  Klin.  Woch.,  1901,  p.  187  ;  ibid., 
I9°3>  P-  J92.  Ewing  and  Strauss,  Proc.  N.  Y.  Path.  Soc.,  vol.  ii.,  p.  152. 


Ewing,  ibid.,  vol.  iii.,  p.    14.      Deutsch,  Cent.  f.  Bakt.   I.  O.,  vol.  xxix., 

E.  661.     Stern,  Deut.  Med.  Woch.,  1901,  p.  135.     Wassermann,  Congr.  f. 
in.  Med.,  1900.     Strube,  Deut.  Med.  Woch.,  1902,  p.  425.     Lenossier  and 


Lemoine,    Sem.    Med.,    1901,    No.   4.     Stern,    Deut.   Med.   Woch.,    1901, 

P-  135- 

Precipitoids,  etc. — Michaelis,  Beit.  z.  Chem.  Phys.,  vol.  iv.,  p.  59.  Ober- 
mayer  and  Pick,  Wien.  Klin.  Woch.,  1903,  No.  22,  and  1904,  p.  265. 
Von  Dungern,  Cent.  f.  Bakt.  I.  O.,  vol.  xxxiv.,  p.  355. 

Kraus's  Reaction. — Wien.  Klin.  Woch.,  1897,  P-  73^  ;  ibid.,  1901,  p.  693. 
Panichi,  Cent.  f.  Bakt.  I.  O.,  vol.  xliii.,  p.  188.  Norris,  Journ.  Inf.  Dis., 
vol.  i.,  p.  463  (with  bibliography).  Hoke,  Wien.  Klin.  Woch.,  vol.  xx., 
p.  347.  Eisler,  Wien.  Klin.  Woch,  vol.  xx.,  p.  377.  Dopter,  Comptes 
Rendus  de  la  Soc.  Biol.,  vol.  lix.,  p.  69.  Smith  and  Reagh,  Journ.  Med. 
Res.,  vol.  x.,  p.  89. 

Serum  Precipitins. — Tchistovitch,  Ann.  Inst.  Past.,  vol.  xiii.,  p.  406. 
Bordet,  ibid.,  p.  225.  Myers,  Cent.  f.  Bakt.  I.  O.,  vol.  xxviii.,  p.  237. 
Wassermann  and  Schutze,  Berlin.  Klin.  Woch.,  1901,  p.  187.  Nuttall, 
Blood  Immunity  and  Blood  Relationship  (Cambridge,  1904),  in  which 
there  is  a  full  bibliography  to  the  date  of  issue.  Uhlenhuth,  Deut.  Med. 
Woch.,  1900,  p.  734.  Michaelis  and  Fleischmann,  Zeit.  f.  Exp.  Path, 
and  Ther.,  vol.  i.,  p.  537.  Von  Dungern,  Cent.  f.  Bakt.  I.  O.,  vol.  xxxiv., 
p.  355.  Obermayer  and  Pick,  Wien.  Klin.  Woch.,  1903,  No.  22  ;  ibid., 
I9°3.  P-  265.  Oppenheimer,  Beit.  z.  Chem.  Phys.,  vol.  iv.,  p.  259. 


BIBLIOGRAPHY  429 

Precipitins  for  Crystalline  Lens. — Uhlenhuth,  Deut.  Med.  Woch.,  1906, 
p.  1244  ;  also  Koch's  Festschrift. 

Practical  Application. — An  excellent  account  of  the  technique  is  given 
by  Welsh  and  Chapman,  Australian  Medical  Gazette,  January  21,  1907- 
See  also  Ewing,  Clinical  Pathology  of  the  Blood,  seond  edition  (Kimpton, 
London).  Graham-Smith  and  Sanger,  Journ.  Hyg.,  vol.  iii.,  pp.  258,  354. 
Buckmaster,  Morphology  of  Blood  (Murray,  1906).  Bruck,  Berlin.  Klin. 
Woch.,  1907,  pp.  793,  1510.  Zebrowski,  C.  R.  Soc.  Biol.,  vol.  Ixii.,  p.  603. 
Uhlenhuth  Deut.  Med.  Woch.,  1906,  p.  1244.  Ziemke,  Deut.  Med.  Woch., 
1 90 1,  pp.  424,  731. 

Deviation  of  Complement. — Neisser  and  Sachs,  Berlin.  Klin.  Woch.,  1905. 
Uhlenhuth,  Deut.  Med.  Woch.,  1906,  p.  1244.  Muir  and  Martin,  Journ.  of 
Hyg.,  1906,  July,  p.  265.  Friedberger,  Deut.  Med.  Woch.,  1906,  p.  578. 

Recognition  of  Foods. — Pniiger,  Arch.  f.  Phys.,  1906,  pp.  465,  540. 
Schmidt,  Bioch.  Zeit.,  vol.  v.,  p.  422.  Uhlenhuth,  Deut.  Med.  Woch., 
1901,  p.  780.  Schutze,  Zeit.  f.  Hyg.,  vol.  xlvii.,  p.  144. 

Recognition  of  Bones. — Schutze,  Deut.  Med.  Woch.,  1903,  p.  62. 


CHAPTER  X 

Metchnikoff's  views  and  experiments  are  fully  set  forth  in  his  "  L'lm- 
munite  dans  les  Maladies  Infecteuses  "  (English  translation  by  Binnie, 
Cambridge  University  Press,  1905),  with  numerous  references,  and  his 
"  Comparative  Pathology  of  Inflammation  "  (translated  by  F.  A.  and  E.  H. 
Starling,  Kegan  Paul,  Trench  and  Co.,  1893).  Buchner,  vol.  xvii.,  p.  138; 
Marchand,  Arch.  Med.  Exp.,  vol.  x.,  p.  253  ;  Massart,  Ann.  Inst.  Past., 
vol.  vi.,  p.  321  ;  Petersson,  Cent.  f.  Bakt.  I.  O.,  vol.  xxxix.,  p.  423  ; 
Savtschenko,  Ann.  Inst.  Past.,  vol.  xvi.,  p.  106  ;  and  numerous  articles 
from  the  French  School  published  in  the  Annales  de  1'Institute  Pasteur, 
Comptes  Rendus  de  la  Soc.  Biol.,  etc.  An  excellent  account  of  the  main 
phenomena  is  given  in  Adami's  article  on  Inflammation  in  Clifford  Allbutt's 
"  System  of  Medicine." 

A  bsorption  of  Tail  of  Tadpole. — Mercier,  Arch.  Zool.  Exper. ,  vol.  v. ,  p.  151. 

Cells  in  Peritoneal  Fluid. — Metchnikoff,  loc.  cit.  Buxton  and  Torrey, 
Journ.  Med.  Res.,  vol.  xv.,  p.  i.  Kanthack  and  Hardy,  Journ.  Phys., 
vol.  xvii.,  p.  81.  Durham,  Journ.  Path,  and  Bact.,  vol.  iv.,  p.  338. 

Phagocytosis  in  the  Lungs. — Briscoe,  Journ.  Path,  and  Bakt.,  1907. 
Baumgarten,  Cent.  f.  Inn.  Med.,  1888,  Zeigler's  Beit.,  1889,  and  Berlin. 
Klin.  Woch.,  1884.  Sanarelli,  Cent.  f.  Bakt.  I.  O.,  vol.  x.,  p.  514.  Kant- 
hack  and  Hardy,  Phil.  Trans.,  1894,  Journ.  of  Phys.,  1894. 

Enterokinase,  etc. — Delezenne,  vide  Levaditi,  L'Immunite. 

Opsonins. — Sir  Almroth  Wright's  researches  have  recently  been  pub- 
lished in  book-form  (Studies  in  Immunization,  Constable,  1909),  to  which 
the  reader  is  referred  for  a  full  account  of  the  main  researches  on  the 
subject.  See  also  the  Practitioner,  special  number,  May,  1908,  and  the 


discussion   on    Phagocytosis,    B.    M.    J.,    November    16,    1907.     See   also 

Rimpau,    Deut.   Med.    Woch.,    1904,   p. 
B.  M.   J.,   1902.     Dean,  Proc.  Roy.  Soc.,    1905,  vol.  Ixxvi.,  p.   506,  and 


Neufeld  and    Rimpau,    Deut.   Med.    Woch.,    1904,   p.    1458.       Leishman, 


May  30,  1907.  Muir  and  Martin,  B.  M.  J.,  1907,  p.  1783.  Noguchi, 
Journ.  Exp.  Med.,  vol.  ix.,  p.  455.  Rosenow,  Journ.  Inf.  Dis.,  vol.  iv., 
p.  285.  Gruber  and  Futaki,  Munch.  Med.  Woch.,  vol.  liii.,  p.  249.  Hektoen 
and  Ruediger,  Journ.  Inf.  Dis.,  vol.  ii.,  p.  128.  Bulloch  and  Atkin,  Proc. 
Roy.  Soc.,  vol.  Ixxiv.,  p.  379.  Neufeld,  Arb.  der  Kais.  Gesundh.,  vol.  xxv., 
p.  164,  and  Berlin.  Klin.  Woch.,  1908,  p.  993.  Lohlein,  Ann.  Inst.  Past., 
vol.  xix.,  p.  647,  and  vol.  xxx.,  p.  939.  Weil,  Cent.  f.  Bakt.  (Ref.),  1908, 

P-  337- 

Technique  of  Opsonin  Estimations,  etc. — Leishman,  B.  M.  J.,  January  n, 
1902.     Wright  and  Douglas,  Proc.  Roy.  Soc.,  vols.  Ixxii.,  Ixxxiii.     Fleming, 


430  BIBLIOGRAPHY 

Practitioner,  May,  1908.  Walker,  R.  E.,  Journ.  Med.  Res.,  vol.  xix., 
p.  237.  Klien,  Bull.  Johns  Hopkins  Hosp.,  1907,  p.  245.  Simon,  Journ. 
Amer.  Med.  Assoc.,  1907,  p.  139.  Hektoen,  Journ.  Inf.  Dis.,  vol.  iii., 
p.  434.  Veitch,  Journ.  Path,  and  Bact.,  January,  1908.  Brown,  Journ. 
Amer.  Med.  Assoc.,  1908.  Morland,  Inaugural  Dissertation  (Bern,  1908). 
Emery,  Clinical  Pathology  and  Bacteriology,  third  edition  (H.  K.  Lewis, 
1908). 

Opsonic  Index  in  Health. — Bulloch,  Trans.  Path.  Soc.,  vol.  Ivi.  Fleming, 
Practitioner,  May,  1908.  Hollister,  quoted  by  Bergey,  Monthly  Cyclop, 
of  Prac.  Med.,  August,  1907.  Urwick,  B.  M.  J.,  1905,  July  22.  Frazer, 
Glas.  Med.  Journ.,  March,  April,  etc. 

Opsonic  Indices  in  Diseases. — See  under  the  appropriate  headings  below. 
Accuracy  of  Opsonic  Determinations. — Greenwood,  Proc.  Roy.  Soc.  Med., 
vol.  ii.,  No.  5,  where  a  full  bibliography  is  given. 

Nature  of  Opsonins. — Crofton,  Journ.  Hyg.,  vol.  v.,  p.  949.  Chapin  and 
Cowie,  Journ.  Med.  Res.,  vol.  xvii.,  p.  213.  Dean,  Proc.  Roy.  Soc.,  1907, 
p.  399.  Levaditi  and  Inman,  Arb.  Kais.  Gesund.,  vol.  xxv.,  p.  164. 
Ledingham,  Proc.  Roy.  Soc.,  1907.  McFarlane,  Journ.  Amer.  Med.  Assoc., 
vol.  xlix.,  p.  1178.  Noguchi,  Journ.  Exp.  Med.,  vol.  ix.,  p.  455.  Simon, 
Journ.  Exp.  Med.,  vol.  ix.,  p.  487.  Eggers,  Journ.  Inf.  Dis.,  vol.  v.,  p.  268. 
Graham,  ibid.,  p.  273.  Bohme,  Munch.  Med.  Woch.,  1908,  p.  1475. 
Neufeld  and  Bickel,  Ar.b.  Kais.  Gesund.,  vol.  xxvii.,  p.  310.  Levaditi  and 
Inman,  C.  R.  Soc.  Biol.,  vol.  Ixii.,  p.  683.  Eggers,  Journ.  Inf.  Dis.,  vol.  v., 
p.  263.  Hektoen  and  Ruediger,  Journ.  Inf.  Dis.,  vol.  ii.,  p.  128.  Hektoen, 
Journ.  Inf.  Dis.,  vol.  iii.,  p. 434.  Browning,  Journ.  Med.  Res.,  vol.  xix.,  p.  201. 
Specificity  of  Opsonins. — Bulloch  and  Western,  Proc.  Roy.  Soc.,  vol. 
Ixxvi.  Simon,  Journ.  Exp.  Med.,  1906,  p.  651.  Muir  and  Martin  (W.  B.  M.), 
B.  M.  J.,  1906,  vol.  ii.,  p.  1783.  Potter,  Ditman,  and  Bradley,  Journ. 
Amer.  Med.  Assoc.,  vol.  xlvii.,  p.  1793.  Russell,  Bull.  Johns  Hopkins 
Hosp.,  1907,  p.  252.  Hektoen,  Journ.  Inf.  Dis.,  vol.  v.,  p.  249.  McFarland 
and  L'Engle,  Journ.  Amer.  Med.  Assoc.,  vol.  xlix.,  p.  1178. 

Thermolability  of  Opsonins. — Wright  and  Douglas,  Proc.  Roy.  Soc., 
vol.  Ixxii.  Wright  and  Reid,  ibid.,  vol.  Ixxvii.  Macdonald,  Studies  in 
Path.  Aberd.  Uni.,  1906.  Rosenow,  Journ.  Inf.  Dis.,  vol.  iii.,  p.  683. 
Muir  and  Martin,  B.  M.  J.,  1906,  vol.  ii.,  p.  1783  ;  and  Proc.  Roy.  Soc., 
vol.  Ixxix.,  p.  187.  Neufeld  and  Hime,  Arb.  Kais.  Gesund.,  vol.  xxv., 
p.  164.  Dean,  B.  M.  J.,  Nov.  16,  1907  (with  an  excellent  general  account 
of  the  subject  to  date).  See  also  under  Nature  of  Opsonins. 

Influence  of  Temperature. — Bulloch  and  Atkins,  Proc.  Roy.  Soc.,  vols. 
Ixxii.  and  Ixxiii.  Ledingham,  ibid.,  1908. 

Influence  of  Source  of  Leucocytes. — Wright  and  Douglas,  Proc.  Roy.  Soc., 
vol.  Ixxiv.  Bulloch  and  Ledingham,  Studies  in  Path.  Univ.  Aberdeen, 
1906.  Fleming,  Practitioner,  May,  1908.  Rosenow,  Journ.  Inf.  Dis., 
vol.  iii.,  p.  683.  Lowenstein,  Zeit.  f.  Hyg.,  vol.  lv.,  p.  429.  Bassett- 
Smith,  Journ.  Hyg.,  1907,  p.  115.  Shattock  and  Dudgeon,  Proc.  Roy. 
Soc.  Med.,  vol.  i.,  No.  6. 

Virulence.— See  Chapter  XIII. 

Influence  of  Salts,  etc. — Wright  and  Reid,  Proc.  Roy.  Soc.,  vol.  Ixxvii. 
Hamburger  and  Hekma,  Biochem.  Zeit.,  vol.  ix.,  pp.  275,  512.  Sellards, 
Journ.  Inf.  Dis.,  1908,  June.  Noguchi,  Journ.  Exp.  Med.,  vol.  ix.,  p.  455. 
Influence  of  Dilution  of  Serum. — Wright  and  Douglas,  Proc.  Roy.  Soc., 
vol.  Ixxii.  Emery,  Trans.  Med.  Chi.  Soc.,  vol.  Ixxxix.  Marshall,  Journ. 
Path.  Bact.,  1908,  p.  378. 

Influence  of  Thickness  of  Bacterial  Emulsion. — Tunnicliffe,  Journ.  Inf. 
Dis.,  1908,  January.  Walker,  Journ.  Med.  Res.,  vol.  xvi.,  p.  521. 

Hcemopsonins. — Neufeld  and  Bickel,  Arb.  Kais.  Gesund.,  vol.  xxvii., 
p.  310.  Neufeld  and  Topfer,  Cent.  f.  Bakt.  I.  O.,  vol.  xxxviii.,  p.  456. 
Barratt,  Wakelin,  .Proc.  Roy.  Soc.,  1905,  p.  524.  Keith,  Proc.  Roy. 
Soc.,  1906. 

Aggressins. — Bail,   O.,  Wien.    Klin.    Woch.,    vol.    xvii.,    p.    846;     ibid., 


BIBLIOGRAPHY  43! 


vol. 
Woch 


xviii.,  p.  428.  Munch.  Med.  Woch.,  1905,  pp.  1212,  1865  ;  Deut.  Med. 
h.,  1905,  p.  1788.  Bail  and  Weil,  Cent.  f.  Bakt.  I.  O.,  vol.  xl.,  p.  371. 
sermann  and  Citron,  Cent.  f.  Bakt.  I.  O.,  vol.  xliii.,  p.  373  ;  and  Deut. 


Wassermann  and  Citron,  Uent.  l.  .tsakt.  l.  u.,  vol.  xlni.,  p.  373  ; 

Hyg.,  vol.  liii.,  p. 
Cent.  f.  Bakt,  vol.  xli.,  p.  230.      Weil,  Deut.  Med.  Woch.,  1906,  p.  382  ; 


Klin.  Woch.,  1905,  p.  1102.     Citron,  Zeit.  f.  Hyg.,  vol.  liii.,  p.  515  ;  ibid., 


ibid.,  Wien.  Klin.  Woch.,  1905,  p.  406;  ibid.,  Arch.  f.  Hyg.,  vol.  liv.,  p.  297  ; 
and  Berlin.  Klin.  Woch.,  1905,  p.  430.  Salus,  Arch.  f.  Hyg.,  vol.  lv., 
P-  335  >'  ibid.,  Wien.  Klin.  Woch.,  vol.  xviii.,  p.  660.  Especially  Lancet, 
August  17  and  24,  1907  (Collected  Studies,  p.  317). 

Vaccine  Treatment. — Wright's  Collected  Studies.  Especially  Lancet, 
August  17  and  24,  1907  (Collected  Studies,  p.  327).  Practitioner,  May, 
1908.  Allen's  Vaccine-Therapy  (H.  K.  Lewis,  1908).  Pfeiffer  and  Fried- 
berger,  Cent.  f.  Bakt.  I.  O.,  vol.  xlvii.,  p.  503.  See  also  under  the  separate 
headings. 

CHAPTER  XI 

Tuberculin  Reaction. — Koch,  Deut.  Med.  Woch.,  1890  and  1891.  Wasser- 
mann and  Bruck,  Deut.  Med.  Woch.,  1906,  p.  449  (in  which  there  is  a  good 
account  of  the  earlier  theories).  (See  also  Chapter  XIV.) 

Modifications  of  Tuberculin  Reaction. — See  under  Tubercle. 

Mallein  Reaction. —  Vide  Jowett's  Blood-Serum  Therapy,  p.  156.  Kraus 
and  Levaditi,  vol.  i. 

Reactions  in  Gonococcal  Infections. — Irons,  Arch.  Int.  Med.,  vol.  i.,  p.  433. 
Difference  in  Reactions  between  Healthy  and  Infected  Persons. — Lawson  and 
Stewart,  Proc.  Med.  Chi.  Soc.,  1905.  See  also  Allen's  Vaccine  Therapy. 

Anaphylaxis  to  Toxins. — Richet,  Comptes  Rendus  Soc.  Biol.,  vol.  Iviii., 
p.  109  ;  Ann.  Inst.  Past.,  vol.  xxi.,  p.  497  ;  and  Comptes  Rendus  Soc.  Biol., 
vol.  Ixii.,  pp.  358,  643.  Goodman,  Journ.  Inf.  Dis.,  vol.  iv.,  p.  509. 

Hyper  sensitiveness  to  Serum. — Arthus,  Comptes  Rendus  Soc.  Biol., 
vol.  lv.,  p.  817.  Nicolle,  Ann.  Inst.  Past.,  vol.  xxi.,  p.  128.  Remlinger, 
Comptes  Rendus  Soc.  Biol.,  vol.  Ixii.,  p.  23. 

Theobald  Smith's  Phenomenon. — Rosenau  and  Anderson,  Journ.  Med. 
Res.,  vol.  xv.,  p.  179  ;  ibid.,  vol.  xvi.,  p.  381  ;  and  Journ.  Amer.  Med. 
Assoc.,  1906,  p.  1007.  Besredka  and  Steinhardt,  Ann.  Inst.  Past.,  vol.  xxi., 
p.  117.  Besredka,  Comptes  Rendus  Soc.  Biol.,  vol.  Ixii.,  p.  477;  ibid., 
vol.  Ixiii.,  p.  294  ;  ibid.,  Ann.  Inst.  Past.,  vol.  xxi.,  p.  950  ;  and  Bull. 
Inst.  Past.,  vol.  vi.,  p.  841.  Gay  and  Southard,  Journ.  Med.  Res.,  vol.  xv., 
p.  143.  Vaughan  and  Wheeler,  Journ.  Inf.  Dis.,  1907,  p.  476.  Otto, 
Munch.  Med.  Woch.,  1907.  Doerr,  Wien.  Klin.  Woch.,  1908.  Gay  and 
Southard,  Journ.  Med.  Res.,  vol.  xviii.,  p.  407.  Weil-Halle  and  Lemaire, 
Comptes  Rendus  Soc.  Biol.,  vol.  Ixiii.,  p.  748.  Lewis,  Journ.  Exp.  Med., 
vol.  x. 

Serum  Disease. — Von  Pirquet  and  Schick,  Die  Serum-Krankheit  (Leipzic 
and  Wien,  1905).  Currie,  Journ.  Hyg.,  vol.  vii.,  p.  35.  Goodall,  Journ. 


Hyg.,  vol.  vii.     Hamburger  and  Moro,  Wien.  Klin.  Woch.,  vol.  xvi.,  p.  445. 

vii.,  p.  807,  and  xx.,  p.  817.     Wic 
and  Rostane,  Bull.  Soc.  Med.  des  Hop.  de  Paris,   1905,  p.  424.     Marfan 


Hamburger  and  Dehne,  ibid.,  vol.  xvii.,  p.  807,  and  xx.,  p.  817.     Widal 


and  Le  Play,  ibid.,  p.  274.  Netter,  Comptes  Rendus  Soc.  Biol.,  vol.  lx., 
p.  279.  Park  and  Throne,  Trans.  Assoc.  Amer.  Phys.,  vol.  xxi.,  p.  259. 
Saunders,  Interstate  Med.  Journ.,  1908,  p.  576. 

CHAPTER  XII 

A  good  general  outline  of  the  subject  may  be  found  in  Pauli's  "  Physical 
Chemistry  in  the  Service  of  Medicine,"  1907,  translated  by  Fischer  (Chap- 
man and  Hall).  See  also  Findlay's  "  Physical  Chemistry  in  Medical  and 
Biological  Science  "  (Longmans,  Green  and  Co.,  1905). 

Biltz,  Zeit.  f.  Phys.  Chem.,  vol.  xlviii.,  p.  615.  Biltz  and  Siebert,  Beitr. 
z.  Exp.  Therap.,  1905,  p.  30.  Field  and  Teague,  Journ.  Exp.  Med.,  vol. 


432  BIBLIOGRAPHY 

viii.,  p.  222  ;  and  vol.  ix.,  p.  86.  Teague  and  Buxton,  Journ.  Exp.  Med., 
vol.  ix.,  p.  254.  Craw,  Proc.  Roy.  Soc.,  vol.  Ixxvi.,  p.  179  ;  and  vol.  Ixxvii., 
p.  311,  and  other  articles.  Bordet,  Ann.  Inst.  Past.,  vol.  xvii.,  p.  161. 
Nernst,  Zeit.  f.  Electrochemie,  vol.  x.,  p.  377.  Girard-Mangin  and  Henri, 
Comptes  Rendus  Soc.  Biol.,  vol.  Ivi.,  p.  541,  and  numerous  other  articles 
in  the  same  periodical  and  in  Comptes  Rendus  Acad.  Sci.  Landsteiner 
and  Stancovic,  Cent.  f.  Bakt.  I.  O.,  vol.  xli.,  p.  108.  Landsteiner  and 
Urlirz,  Cent.  f.  Bakt.  I.  O.,  vol.  xl.,  p.  265.  Flexner  and  Noguchi,  Journ. 
Exp.  Med.,  1906,  p.  547.  Bechhold,  Zeit.  f.  Phys.  Chem.,  vol.  xlviii., 
p.  385.  Neisser,  Cent.  f.  Bakt.  I.  O.,  vol.  xxxvi.,  p.  671.  Neisser  and 
Friedemann,  Munch.  Med.  Woch.,  1904,  p.  465.  Michaelis  and  Fleisch- 
mann,  Zeit.  f.  Exp.  Path.  u.  Ther.,  vol.  i.,  p.  547.  Gengou,  Ann.  Inst. 
Past.,  vol.  xviii.,  p.  678.  Dreyer,  B.  M.  J.,  September  10,  1904. 

Danysz  Effect. — Danysz,  Ann.  Inst.  Past.,  vol.  xvi.,  p.  331.  Jacoby, 
Hoffm.  Beit.,  vol.  iv.,  p.  212.  Sachs,  Cent.  f.  Bakt.  I.  O.,  vol.  xxxvii., 
p.  251.  Craw,  Proc.  Roy.  Soc.,  1905. 

Precipitation  of  Colloids. — Spiro,  Beit.  z.  Chem.  Phys.,  vol.  iv.,  p.  300. 
Perrin,  Comptes  Rendus  Acad.  Sci.,  vol.  cxxxvi.,  p.  564. 

Hcemolysis  by  Silicic  Acid. — Landsteiner  and  Jagic,  Wien.  Klin.  Woch., 
vol.  xvii.,  p.  63. 

CHAPTER  XIII 

Phagocytosis  in  Peritoneum. — Buxton  and  Torrey,  Journ.  Med.  Res., 
vol.  xv.,  p.  5.  Petterson,  Cent.  f.  Bakt.  I.  O.,  vol.  xl.,  p.  537.  Weil,  ibid., 
vol.  xliii.,  p.  190,  and  vol.  xliv.,  p.  164  ;  and  Arch.  f.  Hyg.,  vol.  Ixi.,  p.  293  ; 
Journ.  Inf.  Dis.,  vol.  iv.,  p.  582.  Metchnikoff,  L'lmrrhinite.  Pierallini,  Ann. 
Inst.  Past.,  vol.  xi.,  p.  308.  Wolff,  Berlin.  Klin.  Woch.,  1903,  Nos.  17-20. 

Bacterial  Immunity  in  General. — Metchnikoff,  L'Immunite,  especially 
chapters  vi.  to  x.  Sauerbeck,  Die  Krise  in  der  Immunitatsforschung, 
Folia  Serologica,  vol.  ii.,  p.  i,  with  full  bibliography.  Hahn,  Kolle,  and 
Wassermann's  Handbuch,  Fasc.  xviii.  and  xix.  Cole,  Rufus,  Zeit.  f. 
Hyg.,  vol.  xlvi.,  p.  371.  Kisskalt,  Zeit.  f.  Hyg.,  vol.  xlv.,  p.  i.  Hoke,  Zeit. 
f.  Hyg.,  vol.  xxv.,  p.  197.  Bail,  Arch.  f.  Hyg.,  vol.  Hi.,  p.  272.  Neufeld, 
Arb.  a.  d.  Kais.  Gesundh.,  vol.  xxviii.,  p.  125.  W'erigo,  Ann.  Inst.  Past., 
vol.  viii.  Bail,  Arch.  f.  Hyg.,  vol.  liii.,  p.  272.  Hoke,  Cent.  f.  Bakt.  I.  O., 
vol.  xxxiv.,  p.  693.  Sir  Watson  Cheyne,  Lancet,  June  27,  1908. 

In  Tick  Fever. — Levaditi  and  Manouelian,  Comptes  Rendus  Soc.  Biol., 
vol.  Ixi.,  p.  566,  and  vol.  Ixii.,  pp.  619,  815. 

Virulence. — Walker,  Ainley,  Cent.  f.  Bakt.  I.  O.,  vol.  xxxiii.,  p.  297. 
Shaw,  B.  M.  J.,  1903,  May  9,  p.  1074.  Cohn,  Zeit.  f.  Hyg.,  vol.  xlv.,  p.  61. 
Pfeiffer,  Koch's  Festschrift,  1903.  Stiirtz,  Zeit.  f.  Klin.  Med.,  vol.  Hi., 
p.  422.  Bail,  Wien.  Klin.  Woch.,  vol.  xvii.,  p.  846.  Petterson,  Cent.  f. 
Bakt.  I.  O.,  vol.  xxxviii.,  p.  73.  Steinhardt,  Proc.  N.Y.  Path.  Soc.,  vol.  iv. 
Day,  Journ.  Inf.  Dis.,  1905,  p.  569.  Marshall  and  Knox,  Journ.  Med. 
Res.,  vol.  xv.,  p.  325.  Friedberger,  Cent.  f.  Bakt.  I.  O.,  vol.  xliv.,  p.  32. 
Rosenow,  Journ.  Inf.  Dis.,  vol.  iv.,  p.  285. 

Formation  of  Envelope,  etc. — Metchnikoff,  L'Immunite,  chapter  i. 
Danysz,  Ann.  Inst.  Past.,  vol.  xiv.,  p.  641.  Bordet,  ibid.,  vol.  xi.,  p.  177. 
Gruber  and  Futaki,  Munch.  Med.  Woch.,  1906,  p.  249.  Preis,  Cent.  f. 
Bakt.  I.  O.,  vol.  xliv.,  p.  209.  Bail,  Wien.  Klin.  Woch.,  vol.  xix.,  p.  1278. 
Bail  and  Rubritius,  Cent,  f.  Bakt.  I.  O.,  vol.  xliii.,  p.  641.  Stienon, 
Comptes  Rendus  Soc*  Biol.,  vol.  xii.,  pp.  604,  841. 

CHAPTER  XIV 

Staphylococci ;  Staphylolysin. — Van  de  Velde,  Ann.  Inst.  Past.,  vol.  xv., 
p.  580.  Kraus  and  Clairmont,  Wien.  Klin.  Woch.,  1900.  Neisser  and 
Wechsberg,  Zeit.  f.  Hyg.,  vol.  xxxvi.,  p.  299. 

Leucocidine. — Van  de  Velde,  loc.  cit.  Bail,  Arch.  f.  Hyg.,  vol.  xxxii., 
p.  133.  Neisser  and  Wechsberg,  Munch.  Med.  Woch.,  1902,  p.  1261. 


BIBLIOGRAPHY  433 

Immunity. — Nuttall,  Zeit.  f.  Hyg.,  vol.  iv.,  p.  353.  Wright  and  Windsor, 
Journ.  of  Hyg.,  vol.  ii.,  p.  397.  Andrewes  and  Gordon,  Suppl.  Report 
Med.  Officer  L.G.B.,  1906,  p.  141.  Wright  and  Douglas,  Proc.  Roy.  Soc., 
vol.  Ixxxii.,  and  other  articles  in  Wright's  Collected  Studies. 

Vaccine  Treatment,  Opsonins,  etc. — Wright,  Lancet,  March  29,  1902  ; 
B.  M.  J.,  May  7,  1904,  etc.  Allen's  Vaccine  Therapy.  Chapman  and 
Cowie,  Journ.  Med.  Res.,  vol.  xvii.,  p.  i. 

Streptococcic  Infections;  Streptocolysin. — Besredka,  Ann.  Inst.  Past., 
vol.  x.,  p.  880.  Casagrandi,  quoted  by  Oppenheimer. 

Toxins. — Parascandalo,  Wien.  Klin.,  Woch.  1897,  P-  86 1.  Marmorek, 
Ann.  Inst.  Past.,  vol.  ix.,  p.  593.  Roger,  Comptes  Rendus  Soc.  Biol., 
vol.  xliii.,  p.  538.  Schenk,  Wien.  Klin.  Woch.,  1897,  P-  937-  Breton, 
Comptes  Rendus  Soc.  Biol.,  vol.  Iv.,  p.  886.  Simon,  Cent.  f.  Bakt.  I.  O., 
1903,  pp.  308,  440.  Schlesinger,  Zeit.  f.  Hyg.,  vol.  xliv.,  p.  428. 

Serum  Treatment. — Marmorek,  Ann.  Inst.  Past.,  vol.  ix.,  p.  593  ;  and 
Berlin.  Klin.  Woch.,  1902,  No.  14.  Besredka,  Ann.  Inst.  Past.,  vol.  xviii., 


p.  363.  Aronson,  Deut.  Med.  Woch.,  1903,  p.  439.  Tavel,  Cent.  f.  Bakt. 
I.  O.,  vol.  xxxiii.,  p.  212,  and  vol.  xxxv.,  p.  513.  Neufeld,  Zeit.  f.  Hyg., 
vol.  xliv.,  p.  161.  Simon,  Cent.  f.  Bakt.  I.  O.,  vol.  xxxv.,  pp.  308,  440. 


Bordet,  Ann.  Inst.  Past.,  1897,  p.  177.  Wright,  Clin.  Journ.,  1906,  p.  78. 
Neufeld,  Zeit.  f.  Hyg.,  vol.  xliv.,  p.  161.  Sommerfeld,  Cent.  f.  Bakt.  I.  O., 
vol.  xxxiii.,  p.  722. 

Vaccine  Treatment. — Wright,  Practitioner,  May,  1908  ;  and  Lancet, 
August  24,  1907.  Douglas,  Lancet,  February  23,  1907.  Crowe  and 
Wynn,  B.  M.  J.,  August  8,  1908,  p.  303.  Sutcliffe  and  Bayley,  Lancet, 
August  10,  1907.  Tunnicliffe,  Journ.  Inf.  Dis.,  vol.  v.,  p.  268.  Banks, 
Journ.  Path.  Bact.,  1908,  p.  113. 

Pneumococcic  Infections:  Toxin. — Klemperer,  Berlin.  Klin.  Woch,  1891, 
and  Zeit.  f.  Klin.  Med.,  vol.  xx.,  p.  165.  Washbourn,  Journ.  Path.  Bact., 
vol.  iii.,  p.  214.  Isaeff,  Ann.  Inst.  Past.,  vol.  vii.,  p.  259.  Casagrandi, 
quoted  by  Oppenheimer.  Mennes,  Zeit.  f.  Hyg.,  vol.  xxv.,  p.  413.  Carnot 
and  Fournier,  Arch.  Med.  Exp.,  1900,  p.  357. 

Serum  Treatment. — Washbourn,  B.  M.  J.,  February  27,  1897,  p.  510  ; 
and  with  Eyre,  ibid.,  1899,  p.  1247  ;  and  Journ.  Path,  and  Bact.,  vol.  v., 
p.  13.  Eyre,  vide  infra.  Pane,  Cent.  f.  Bakt.  I.  O.,  vol.  xxi.,  p.  664. 
Knauth,  Deut.  Med.  Woch.,  1905,  p.  452.  Castresana,  Rev.  de  Ther., 
1905,  No.  1 8.  Tyler,  Journ.  Amer.  Med.  Assoc.,  1901,  p.  1540.  Mennes, 
vide  supra. 

Vaccine  Therapy,  Opsonins,  etc. — MacDonald,  Path.  Studies,  Univer. 
Aberdeen.  Eyre,  Lancet,  February  22,  1908.  Neufeld  and  Rimpau,  Zeit. 
f.  Hyg.,  vol.  li.,  p.  283.  Graham,  Journ.  Inf.  Dis.,  vol.  v.,  p.  273.  Butler 
Harris,  Practitioner,  May,  1908.  Briscoe  and  Williams,  ibid. 

Gonococcic  Infections;  Toxin.  —  Wassermann,  Berlin.  Klin.  Woch., 
1897,  P-  685  ;  and  Zeit.  f.  Hyg.,  vol.  xxvii.,  p.  298.  Christmas,  Ann. 
Inst.  Past.,  vol.  xi.,  p.  609.  Nicolaysen,  Cent.  f.  Bakt.  I.  O.,  vol.  xxii., 

P-  305- 

Serum  Diagnosis,  Immunity,  etc. — Torrey,  Journ.  Med.  Res.,  vol.- xvii., 
p.  347,  and  vol.  xix.,  p.  471.  Teague  and  Torrey,  ibid.,  vol.  xvii.,  p.  223. 
Meakins,  Johns  Hopkins  Hosp.  Bull.,  1907,  p.  255.  Ricketts,  Infection 
and  Immunity.  Bruckner  and  Christeanu,  Comptes  Rendus  Soc.  Biol., 
vol.  Ix.,  May,  June.  Miiller  and  Oppenheim,  Wien.  Klin.  Woch.,  vol.  xix., 
p.  894.  Bruck,  Deut.  Med.  Woch.,  1906,  p.  1368.  Vannod,  ibid.,  1906, 
p.  1984.  Rogers,  Cent.  f.  Bakt.  I.  O.,  vol.  xxxix.,  p.  279. 

Vaccine  Treatment,  Opsonins,  etc. — Wright,  Lancet,  August  17  and  24, 
1907.  Allen,  Vaccine  Therapy.  Rons,  Arch.  Int.  Med.,  vol.  i.,  p.  433. 
Cole  and  Meakins,  Bull.  Johns  Hopkins  Hosp.,  1907,  p.  223.  Butler  and 
Long,  Journ.  Amer.  Med.  Assoc.,  1908,  p.  744. 

Meningococcic  Infections :  Toxins,  Immunity. — Lepierre,  Journ.  Phys. 
et  Path.  Gen.,  vol.  v.,  p.  547.  Houston  and  Rankin,  B.  M.  J.,  Novem- 
ber 16,  1907.  Davis,  Journ.  Inf.  Dis.,  vol.  ii. 

28 


434  BIBLIOGRAPHY 

Agglutination. — Kutscher,  Deut.  Med.  Woch.,  1906,  p.  1849.  Alice 
Taylor,  Lancet,  July  6,  1907. 

Serum  Treatment. — Kolle  and  Wassermann,  Deut.  Med.  Woch.,  1906, 
p.  609.  Ruppel,  ib'id.,  1906,  p.  1366.  Markl,  Cent.  f.  Bakt.,  vol.  xliii., 
p.  95.  Levy,  Deut.  Med.  Woch.,  1908,  p.  139.  Emmett  Holt,  B.  M.  J., 
October  31,  1908.  Flexner  and  Jobling,  Journ.  Exp.  Med.,  1908,  pp.  141, 
690.  Jochman,  Deut.  Med.  Woch,  1906,  p.  788.  Meyer  and  Ruppel, 
Mediz.  Klin.,  1907,  No.  4,  and  Cent.  f.  Bakt.  I.  O.,  1907.  Wassermann, 
Deut.  Med.  Woch.,  1907,  p.  1585. 

Vaccine  Therapy,  Opsonins,  etc. — McKenzie  and  Martin,  ibid.,  October  31, 
1908,  and  Journ.  Bact.,  1908,  vol.  xii.,  p.  539.  Davis,  Journ.  Inf.  Dis., 
vol.  ii.,  and  vol.  iv.,  p.  538.  Houston,  B.  M.  J.,  November  16,  1907. 
Mackenzie,  ibid.,  June  15,  1907. 

Malta  Fever. — Wright  and  Smith,  Lancet,  March  6,  1897.  Birt  and 
Lamb,  Lancet,  September  9,  1899.  Eyre,  J.  W.  H.,  and  Shaw,  H.  E.  A., 
Report  of  Royal  Society's  Comm.  on  Med.  Fever,  part  v.  Bassett-Smith, 
Journ.  Trop.  Med.  and  Hyg.,  1907,  and  Journ.  Hyg.,  vol.  vii.,  p.  115. 
Eyre,  Lancet,  1908,  June  13,  20,  and  27. 

Tubercle  ;  Tuberculin  Reaction. — Koch,  Deut.  Med.  Woch.,  1890,  p.  1028, 
and  1891,  pp.  101,  1188.  (See  also  1890,  p.  1053  et  seq.)  Babes,  Zeit.  f, 
Hyg.,  vol.  xxxii.  Marmorek,  Comptes  Rendus  Soc.  Biol.,  1903,  p.  1650. 
Ehrlich,  Inter.  Kong.  f.  Hyg.,  1900.  Trudeau,  Baldwin,  and  Kinghorn, 
Journ.  Med.  Res.,  vol.  xii.,  p.  169.  Weil  and  Nakajama,  Munch.  Med. 
Woch.,  1906,  p.  1001.  Cohn,  Berlin.  Klin.  Woch.,  1908,  p.  1309.  Richet, 
Comptes  Rendus  Soc.  Biol.,  1905,  p.  109.  Citron,  Berlin.  Klin.  Woch., 
I9°7>  P-  TI35-  Marmorek,  Lancet,  December  12,  1903  (in  diagnosis 
especially).  Arloing,  Journ.  de  Phys.  et  Path.  General,  1903,  p.  677. 
V.  Bergmann,  Deut.  Med.  Woch.,  1890,  p.  1073,  and  Munch.  Med.  Woch., 
p.  824.  Beck,  Arch.  f.  Kinderheilkunde,  1903,  p.  i.  Lowenstein,  Kraus, 
and  Levaditi,  vol.  i.,  p.  1019  (with  full  bibliography).  Lingelsheim, 
Deut.  Med.  Woch.,  1898,  p.  583.  Armand-Delille,  These  de  Paris,  1903, 
abs.  in  Bull.  Inst.  Past.,  vol.  ii.,  p.  73.  For  an  account  of  the  main  forms 
of  the  tuberculins,  see  Allen's  Vaccine  Therapy,  and  Gamble,  Pharma- 
ceutical Journal,  February  16,  1909. 

Tuberculinin  Treatment. — Koch,  Deut.  Med.  Woch.,  1891,  No.  3.  Denys, 
Comptes  Rendus  Congr.  Tuberc.,  1898,  p.  497.  Petruschky,  Berlin.  Klin. 
Woch.,  1899,  pp.  1 1 20,  1141.  Moller  and  Kayserling,  Zeit.  f.  Tuberkulose, 

1902,  p.  4.     Bandelier,  Deut.  Med.  Woch.,  1898,  p.  798  ;  ibid.,  Zeit.  f.  Hyg., 
vol.  xliii.,  p.  335.     Pardoe,  Lancet,  December  16,  1905.     Spengler,  Deut. 
Med.  Woch.,  1897,  No.  36.     Lowenstein  and  Rappoport,  Zeit.  f.  Tuber- 
kulose, vol.  v.,  p.  6.     Stone  and  Miller,  Medical  Record,  March  28,  1908. 
Hamburger,  Munch.  Med.  Woch.,  vol.  Iv.,  p.  1741. 

Serum  Treatment. — Maragliano,  Berlin.  Klin.  Woch.,  1903,  pp.  563,  593. 
Marmorek,  Berlin.  Klin.  Woch,  1903,  p.  1108. 

Tuberculin  in  Immunization  of  Animals. — Macfadyen,  Journ.  Comp.  Path, 
and  Therap.,  1901,  p.  136;  1902,  p.  60.  Behring,  Berlin.  Klin.  Woch., 

1903,  p.  233,  and  Deut.  Med.  Woch.,  1903,  p.  689.     Behring,  Romer,  and 
Ruppel,  Beitr.  zur  Exp.  Therap.,  vol.  v.     Pearson  and  Gilliland,  Univ. 
Penns.  Med.  Bull.,  vol.  xviii.,  No.  2.     Neufeld,  Deut.  Med.  Woch.,   1904. 
Baumgarten,  Berlin.   Klin.  Woch.,    1905,  No.   3.     Vallee  and  Rossignol, 
Bull.  Soc.  Med.  Vet.  Pratique,  1906,  p.  39. 

Cuti-Re action. — V.  Pirquet,  Klin.  Studien  iiber  Vaccination  and  Vac- 
cinale  Allergic  (Deut.  Wien.,  1907)  ;  Berlin.  Klin.  Woch.,  1907.  Vallee, 
Comptes  Rendus  Acad.  des  Science,  1907,  No.  22.  Ferrand  and  Lemaire, 
La  Presse  Medicale,  1907,  p.  617.  Dufour,  Bull.  Soc.  Med.  Hop.  de  Paris, 
1907.  Engel  and  Bauer,  Berlin.  Klin.  Woch.,  1907,  p.  1169.  Lignieres, 
Bull.  Soc.  Cent.  Med.  Vet.,  1907,  p.  517.  Wolff-Eisner  and  Teichman, 
Berlin.  Klin.  Woch.,  1908,  p.  65. 

Ophthalmo-Reaction. — Wolff-Eisner,  Berlin.  Klin.  Woch.,  1907.  Cal- 
mette,  Comptes  Rendus  Acad.  des  Sciences,  1907.  Vallee,  ibid.  Moro 


BIBLIOGRAPHY  435 

and  Dagonoff,  Wien.  Klin.  Woch.,  1907,  August.  Calmette,  La  Clinique, 
August,  1907.  Chantemesse,  Comptes  Rendus  Acad.  de  Med.,  July  20, 
1907.  Deut.  Med.  Woch.,  September  26,  1907. 

Vaccine  Treatment. —  Vide  numerous  articles  by  Wright  and  his  fellow- 
workers  (in  his  Collected  Studies),  especially  Clinical  Journal,  Novem- 
ber 9,  1904.  Trans.  Med.  Chi.  Soc.,  vol.  Ixxxix.,  and  the  succeeding 
articles  in  the  discussion,  Lancet,  August  17  and  24,  1907.  Reyn  and 
Peterson,  Lancet,  April  4,  1908.  Latham,  Spitta,  and  Inman,  Proc.  Roy. 
Soc.  Med.,  April,  1908.  Torton,  International  Clinics  (eighteenth  series), 
vol.  ii.,  p.  23.  Riviere,  B.  M.  J.,  October  26,  1907.  Whitfield,  Practitioner, 
May,  1908.  Briscoe  and  Williams,  ibid.  Allen,  Vaccine  Therapy.  Car- 
malt  Jones,  Science  Progress,  April,  1909.  Patterson,  Lancet,  Janu- 
ary 25,  1908.  Inman,  ibid. 

Typhoid  Fevsr  ;  Toxin. — Chantemesse,  Prog.  Med.,  1898,  p.  245  ;  Deut. 
Med.  Woch.,  1907,  p.  1572.  Presse  Med.,  1904,  p.  681.  Macfadyen  and 
Rowland,  Proc.  Roy.  Soc.,  vol.  Ixxi.,  p.  77.  Conradi,  Deut.  Med.  Woch., 
1903,  p.  26.  Pfeiffer  and  Kolle,  Zeit.  f.  Hyg.,  vol.  xxi.,  p.  203.  Besredka, 
Ann.  Inst.  Past.,  vol.  xx.,  pp.  149,  304.  Neisser  and  Shiga,  Deut.  Med. 
Woch.,  1903,  p.  6r. 

Immunity,  Bactericidal  Power  of  Blood,  Opsonins,  etc. — Leishman,  Jour. 
R.  A.  M.  C.,  1907.  Evans,  Laming,  Journ.  Path.  Bact.,  1904,  p.  42. 
Shiga,  Berlin.  Klin.  Woch.,  1904,  p.  79.  Richardson,  Journ.  Med.  Res., 
vol.  xiii.  Wright,  Lancet,  September  14,  1901.  Harrison,  Journ.  R.  A. 
M.  C.,  1907,  p.  472.  Stern  and  Korte,  Berlin.  Klin.  Woch.,  1904.  Klien, 
Johns  Hopkins  Hosp.  Bull.,  1907,  p.  245.  Neufeld  and  Kuhn,  Arb.  a.  d. 
K.  Gesundh.,  vol.  xxv.,  p.  164. 

Vaccine  Treatment  (Prophylactic). — Wright,  Short  Treatise  on  Anti- 
Typhoid  Inoculation  (Constable,  1904)  ;  ibid.,  Lancet,  September  6,  1902  ; 
ibid.,  B.  M.  J.,  October  10,  1903.  Luxmore,  Journ.  R.  A.  M.  C.,  1907.  A 
good  account  of  the  subject  is  by  Netter,  Bull.  Inst.  Past.,  vol.  iv.,  pp.  873, 
921,  969,  and  1024.  Shiga,  Berlin.  Klin.  Woch.,  1904,  p.  78.  Friedberger 
and  Moreschi,  Deut.  Med.  Woch.,  1906,  p.  1986. 

Curative. — Richardson,  Boston  Med.  and  Surg.  Journ.,  vol.  Ivii.,  p.  449. 
Cholera  :  Toxin. — Wassermann,  Zeit.  f.  Hyg.,  vol.  xiv.,  p.  35.  Westbrook 
Ann.  Inst.  Past.,  vol.  viii.,  p.  318.  Pfeiffer,  Zeit.  f.  Hyg.,  vol.  xi.,  p.  373, 
and  vol.  xvi.,  p.  268,  and  vol.  xx.,  p.  198.  Metchnikoff,  Roux,  and  Tau- 
relli  Salimbeni,  Ann.  Inst.  Past.,  vol.  x.,  p.  257.  Kraus,  Wien.  Klin. 
Woch.,  vol.  xix.,  p.  655.  Brau  and  Demei,  Ann.  Inst.  Past.,  vol.  xx., 
p.  578.  Macfadyen,  Cent.  f.  Bakt.,  vol.  xlii.,  p.  365. 

Serum  Treatment. — Kraus,  Wien.  Klin.  Woch.,  1909,  No.  2.  Macfadyen, 
Lancet,  August  25,  1906. 

Vaccine  Prophylaxis. — Haffkine,  Bull.  Inst.  Past.,  vol.  iv.,  pp.  697,  737. 
Fischera,  Cent.  f.  Bakt.  I.  O.,  vol.  xli.,  pp.  576,  671,  and  771  (with  full 
bibliography). 

Plague  :  Prophylaxis. — Haffkine,  B.  M.  J.,  June  12,  1897  '<  ibid.,  B.  M.  J., 
September  24,  1898  ;  ibid.,  Proc.  Roy.  Soc.,  1899.,  vol.  Ixv.  ;  ibid.,  Gov. 
Central  Press,  1900,  1903,  1904.  Burch,  N.  Y.  Med.  Journ.,  September, 
1902.  Forsyth,  Lancet,  December  12,  1903.  Lustig  and  Galeotti, 
B.  M.  J.,  October  9  and  November  27,  1897.  Bannerman,  Cent.  f.  Bakt. 
I.  O.,  vol.  xxix.,  p.  857.  Kolle  and  Otto,  Deut.  Med.  Woch.,  1904,  p.  493. 
Lustig  and  Galeotti,  Deut.  Med.  Woch.,  1897,  P-  227- 

Sero-Therapy. — Yersin,  Ann.  Inst.  Past.,  vol.  xi.,  p.  8-1.  Metchnikoff, 
ibid.,  vol.  xi.,  p.  737.  Zabolotny,  ibid.,  vol.  xiii.,  p.  833.  Calmette  and 
Salimbeni,  ibid.,  vol.  xiii.,  p.  865.  Dugardin-Beaumetz,  Bull.  Inst.  Past., 
1906,  p.  453.  Choksy,  Report  on  Treatment  of  Plague,  Bombay,  1906, 
and  Lancet,  1900,  p.  291.  Clemow,  Lancet,  May  6,  1899,  p.  1212.  Cairns, 
Lancet,  1903,  May  9.  Symmers,  Cent.  f.  Bakt.  I.  O.,  vol.  xxv.,  p.  460. 
Markl,  Zeit.  f.  Hyg.,  vol.  xlii.,  p.  244. 

Glanders  :  Immunity. — Nicolle,  Ann.  Inst.  Past.,  vol.  xx.,  pp.  625,  698, 
and  801  (especially  p.  828).  Kleine,  Zeit.  f.  Hyg.,  vol.  xliv.,  p.  183. 

28—2 


436  BIBLIOGRAPHY 

Mallein. — The  directions  given  at  the  Royal  Veterinary  College,  London, 
are  given  in  Hewlett's  Serum-Therapy.  See  also  Jowett's  Blood -Serum 
Therapy. 

The  only  full  account  of  the  subject  is  in  Kraus  and  Levaditi,  vol.  i., 
p.  1090  (Wladimoroff). 

Agglutination. — Bonome,  Cent.  f.  Bakt.  I.  O.,  vol.  xxxviii.,  p.  60 1. 
Heanly,  Lancet,  February^,  1904,  p.  364.  Feodorowsky,  Bull.  Inst. 
Past.,  vol.  ii.,  p.  127. 

Dysentery  :  Toxin. — Rosenthal,  Deut.  Med.  Woch.,  1904,  p.  235.  Todd, 
,/  Journ.  of  Hyg.,  vol.  iv.,  p.  480.  Conradi,  Deut.  Med.  Woch.,  1903,  p.  26. 
Ludke,  Berlin.  Klin.  Woch.,  1906,  pp.  3,  54.  Besredka,  Ann.  Inst.  Past., 
vol.  xx.,  p.  304.  Neisser  and  Shiga,  Deut.  Med.  Woch.,  1903,  p.  61. 

Serum. — Kruse,  Deut.  Med.  Woch.,  1903,  pp.  6,  49.  Shiga,  Cent.  f. 
Bakt.  I.  O.,  1903,  No.  7  ;  ibid.,  Deut.  Med.  Woch.,  1901,  pp.  744, 
765,  and  783  ;  ibid.,  Zeit.  f.  Hyg.,  vol.  xli.,  p.  355  (in  Ehrlich's  Collected 
Studies),  and  vol.  lx.,  p.  75.  Vallard  and  Dopter,  Ann.  Inst.  Past.,  vol.  xx., 
p.  321.  Flexner,  Bull.  Johns  Hopkins  Hosp.,  vol.  xi.,  p.  231.  Besredka, 
vide  supra.  Doerr  in  Kraus  and  Levaditi's  Hardbuch  (with  bibliography). 
Coyne  and  Auche,  Comptes  Rendus  Soc.  Biol.,  vol.  Ixiv.,  p.  829.  Ruffer 
and  Willmore,  B.  M.  J.,  October  17,  1908,  vol.  ii.,  p.  1176.  Heller,  Cent, 
f.  Bakt.  I.  O.,  vol.  xlii.,  p.  30. 

Vaccine  Treatment. — Shiga,  Cent.  f.  Bakt.  I.  O.,  vol.  xxxiv.,  p.  392. 
Forster,  Indian  Med.  Gaz.,  1907,  p.  201  (quoted  by  Allen).  Newman, 
Lancet,  May  16,  1908,  p.  1410.  Kolle  and  Strong,  Deut.  Med.  Woch., 
1906,  p.  413. 

Anthrax:  Toxin. — Conradi,  Zeit.  f.  Hyg.,  vol.  xxxi.,  p.  287  (with  full 
bibliography  to  date). 

Immunity,  Serum  Reactions,  etc. — Sobernheim,  Berlin.  Klin.  Woch., 
1897,  p.  910  ;  ibid.,  Zeit.  f.  Hyg.,  1899,  p.  891.  Bail,  Cent.  f.  Bakt.  I.  O., 
vol.  xxvii.,  p.  10  ;  ibid.,  vol.  xxxiii.,  pp.  343,  610  ;  vol.  xxxvi.,  pp.  266, 
287  ;  vol.  xxxvii.,  p.  270.  Bail  and  Petterson,  ibid.,  vol.  xxxiii.,  p.  756, 
and  vol.  xxxiv.,  pp.  450,  540.  Gengou,  Ann.  Inst.  Past.,  vol.  xiii.,  p.  642. 
Hektoen,  Journ.  Inf.  Dis.,  vol.  iii.,  p  103.  Horton,  ibid.,  vol.  iii.,  p.  no. 
Ascoli,  Zeit.  f.  Hyg.,  vol.  Iv.,  p.  44.  Bandi,  Cent.  f.  Bakt.,  vol.  xxxvii., 
p.  464.  Gruber  and  Futaki,  Deut.  Med.  Woch.,  1906,  p.  1589.  Cler, 
Arch.  Sc.  Med.,  vol.  xxix.,  1905. 

Serum  Treatment. — Legge,  Lancet,  March  25,  1905,  in  which  a  good 
account  of  the  subject  and  the  more  important  references  are  given. 

Diphtheria:  Immunization  to  the  Bacilli. — Bandi,  Cent.  f.  Bakt.  I.  O., 
vol.  xxxiii.,  p.  535.  Rist,  Comptes  Rendus  Soc.  Biol.,  1903,  p.  978. 
Lipstein,  Cent.  f.  Bakt.  I.  O.,  vol.  xxxiii.,  p.  305. 

Opsonic  Action. — Tunnicliffe,  Journ.  Inf.  Dis.,  vol.  v.  Reque,  ibid., 
vol.  iii.,  p.  441. 

No  literature  concerning  the  use  of  diphtheria  antitoxin  need  be  given. 

Tetanus. — A  full  account  of  the  toxin  is  given  in  Oppenheimer,  with  full 
bibliography. 

Action  on  the  Nervous  System. — Gumprecht,  Deut.  Med.  Woch.,  1894, 
p.  546.  Meyer  and  Ransom,  Arch.  Exp.  Path.,  vol.  xlix.,  p.  369.  Marie 
and  Morax,  Ann.  Inst.  Past.,  vol.  xvi.,  p.  818,  and  vol.  xvii.,  p.  335.  Roux 
and  Borrel,  ibid.,  vol.  xii.,  p.  225.  Vaillard  and  Vincent,  ibid.,  vol.  v., 
p.  i.  Marie,  Bull.  Inst.  Past.,  vol.  i.,  p.  633.  Fletcher,  Brain,  1903, 

P-  383- 

Immunity. —  Vide  Metchnikoff,  L'Immunite,  especially  p.  179  (English 
edition,  p.  169)  and  p.  412  (p.  392).  In  the  same  work  much  information 
will  be  found  regarding  the  action  of  tetanus  toxin  on  different  animals. 

Antitoxin. —  Vide  Hewlett's  Serum-Therapy,  where  the  process  of  manu- 
facture is  given. 

Local  Application  of  Antitoxin. — Calmette,  Comptes  Rendus  Acad.  Sci., 
vol.  cxxxvi.,  p.  1150.  £ 

Syphilis. — The  literature  of  the  serum  diagnosis  of  syphilis  has  already 


BIBLIOGRAPHY  437 

assumed  formidable  proportions.  Wassermann,  Neisser,  Bruck,  and 
Schucht,  Zeit.  f.  Hyg.,  vol.  lv.,  p.  451.  Wassermann,  Berl.  Klin.  Woch., 
I9O7.  P-  1599-  Wassermann  and  Plaut,  Deut.  Med.  Woch.,  1906,  p.  1769. 
Wassermann  and  Meier,  ibid.,  1907,  p.  1287.  Neisser,  Bruck,  and  Schucht, 
Deut.  Med.  Woch.,  1906,  p.  1937.  Bruck  and  Stern,  ibid.,  1908,  p.  401. 
Schutze,  Berlin  Klin.  Woch.,  1907,  p.  126.  Levaditi  and  Marie,  Comptes 
Rendus  Soc.  Biol.,  vol.  Ixii.,  p.  872.  Levaditi  and  Yamanouchi,  ibid., 
vol.  Ixiii.,  p.  740,  and  vol.  Ixiv.,  pp.  275,  349,  and  720.  Marie,  Levaditi, 
and  Yamanouchi,  ibid.,  p.  169.  Citron,  Berlin.  Klin.  Woch.,  1907,  p.  1370. 
Michaelis,  ibid.  Meier,  ibid.,  p.  1636.  Weil  and  Braun,  Berlin.  Klin. 
Woch.,  1907,  p.  1570,  and  Wien.  Klin.  Woch.,  1908,  p.  151.  Klausner, 
Wien.  Klin.  Woch.,  1908,  p.  214.  Landsteiner,  Miller  and,  Potzl,  Wien. 
Klin.  Woch.,  1907.  Forges  and  Meier,  Berlin.  Klin.  Woch.,  1908,  p.  731. 
Elias,  Neubauer,  Forges,  and  Salmon,  Wien.  Klin.  Woch.,  1908,  p.  748. 
Simplified  forms  of  technique  are  given  by  Noguchi,  Journ.  Exp.  Med., 
1909,  p.  392,  and  Caulfeild,  Journ.  Med.  Res.,  1908,  p.  507. 

Rabies. — -An  excellent  account  of  modern  views  on  the  immunity  to 
rabies  is  given  by  Marie,  Bull.  Inst.  Past.,  vol.  vi.,  1908,  pp.  705  and  753. 

See  also  Schneder,  Zeit.  f.  Hyg.,  vol.  xlii.,  p.  362.  Remlinger,  Bull. 
Inst.  Past.,  vol.  ii.,  pp.  753  and  792. 


INDEX  OF  AUTHORITIES  CITED 


ABEL,  1 18,  173 
Albarran,  193 
Allen,  367,  370,  384 
Amos,  193,  209 
Anderson,  312 
Andrewes,  293,  359 
Armand-Delille,  170,  196 
Arrhenius,  80,  83  et  seq.,  119 
Arthus,  311 
Arzt,  417 
Ascoli,  193,  233 
Atkinson,  63,  67 
Axenfeld,  366 
Ayer,  188,  190 


Bail,  220,  291,  292,  308,  340,  406 

Bannerman,  404 

Bassenge,  392 

Bassett-Smith,  291,  375 

Baumgarten,  250 

Bechhold,  217 

Beebe,  192 

Bearing,  von,  61,  132,  379 

Bergengriin,  247 

Bernard,  193 

Besredka,  28,  1 13,  172,  190,  242,  314, 

393 

Bickel,  284 
Bier,  31 
Bierry,  192 

Biltz,  90,  217,  324,  327,  328 
Blum,  no 
Blumenthal,  106 
Bolton,  -.94 
Bordet,  88,   153,  168,    180,  209,  212, 

219,  228,  299,  322 
Bousfield,  374 
Bram  Pusey,  197 
Bredig,  320 
Brieger,  54,  61,  411 
Briscoe,  180,  248,  286,  333,  374 
Brodie,  70 
Browning,  286 
Briick,  169,  285,  306 
Brunton,  Sir  L.,  224 
Buchner,  54,  58,  86,  179 


Bulloch,  127,  139,  1 80,  184,  259,  269 

270,  286,  289,  290,  348,  378 
Buxton,  215,  352 


Cairns,  402 

Calmette,  70,  no,  122,   199,  3°3, 

393-  403.  4T3 
Casagrandi,  365 
Castellani,  217 
Cattani,  334 
Centanni,  196,  418 
Chantemesse,  304.  391 
Chapin,  283 
Charrin,  193,  204 
Chatenay,  112 
Chauveau,  18,  35 
Cherry,  48,  70,  329 
Choksy,  403 
Citron,  293 
Cler,  407 
Cohn,  54,  411 
Cole,  352 
Collins,  220 
Conradi,  181,  396,  405 
Corpechot,  193,  197 
Courmont,  107 
Cowie,  283 

Crendiropoulo,  209,  211,  214 
Crofton,  283 
Currie,  309 

D 

Danysz,  327 
Davis,  372 
Dean,  263,  283 
Delaware,  193 
Delbriick,  155 
Delecarde,  122 
Delezenne,  182,  196,  256 
Denys,  179,  257 
Descatello,  224 
Dineur,  211 
Dmitrevsky,  108,  127 
Doerr,  293 
Douglas,  257,  286 
Doyon,  107 
Dreyer,  87,  216,  324 


439 


440 


INDEX    OF    AUTHORITIES    CITED 


Duclaux,  54 

Dudgeon,  262 

Dungern,    von,    159,    192,    230,   327, 

39i 
Durham,  204 


Ehrlich,  16,  33,  45,  69  et  seq.,  87,  94, 

105,  125,  142  et  seq.,  200 
Eisenberg,  216,  230,  343 
Eisenzimmer,  418 
Emden,  von,  214 
Ewing,  235 
Eyre,  14,  34,  266,  288,  365,  366,  374 


j.' 

Falloise,  182,  183 

Ferran,  400 

Field,  320,  329 

Figari,  193 

Flexner,  155,  373 

Forner,  418 

Forster,  397 

Frankel,  207 

Friedberger,  162,  171,  281,  392 

Friedemann,  217 

Frouin,  207 

G 

Gay,  171,  172,  188,  190,  223,  312 

Girard-Mangin,  326,  329 

Gengou,  153,  169,  182 

Golovine,  197 

Goodall,  316 

Goodman,  132,  310 

Gordon,  359 

Gruber,  204,  208,  211 

Guedini,  170 

Guldberg,  81 

Guseff,  180 

H 

Haffkine,  401,  403 
Hahn,  182 

Hamburger,  297,  316 
Hankin,  54,  179 
Hardy,  252,  320 
Harris,  55 
Harris,  Butler,  395 
Hartoch,  416 
Hekma,  297 
Hektoen,  222 
Henri,  326,  329 
Herter,  115 
Hewlett,  198 
Hime,  285 
Hoffmeister,  155 
Hogyes,  419 
Hoke,  231,  341 
Houston,  371,  372,  373 


Ignowtowsky,  44 
Inman,  268 
Irons,  304 
Isaeff,  204,  364 


J 


acobi,  55 

agic,  326 

enner,  20 

ex-Blake,  216,  324 

ochmann,  373 

ones,  Wharton,  224 
Joos,  215 
Jungano,  196 

K 

Kanthack,  70,  184,  244,  252,  334 

King,  132 

Kirschbruch,  219 

Kitashima,  61 

Klein,  188 

Klemperer,  364 

Klien,  263 

Knorr,  73,  133 

Koch,  300,  305 

Kolle,  373,  392 

Korschun,  181 

Kossel,  70 

Kraus,  209,  226 

Kruse,  397 

Kutscher,  372 

Kyes,  155 


Lamb,  232 

Lambotte,  181 

Landsteiner,  190,  220,  222,  326,  417 

Lastschenko,  181,  184 

Laubry,  170 

Lazar,  184 

Leclef,  257 

Ledingham,  128,  259,  286,  295,  416 

Leishman,  261,  284 

Le  Play,  193,  197 

Levaditi,  180,  183,  286,  335,  336,  417 

Levy,  373 

Liepmann,  195 

Lignieres,  387 

Lindemann,  193 

Lingelsheim,  von,  379 

Lipstein,  409 

Loffler,  173 

Longcope,  180 

Lubarsch,  139,  183 

Lustig,  403 

M 

Macdonald,  265,  282,  365 
Macfadyen,  58,  181,  390,  396,  399 
Mackenzie,  373 


INDEX    OF   AUTHORITIES    CITED 


44I 


Madsen,  41,  76,  80,  82,  83  et  stq.,  86, 

93.  H9 

Malvoz,  211,  406 

Manouelian,  335 

Maragliano,  383 

Marenghi,  71 

Marie,  419 

Markl,  257,  259,  402 

Marmorek,  17,  306,  583 

Martin,  282,  373 

Martin,  S.,  54 

Marx,  184,  214 

Massart,  299 

McClintock.  132 

McFarland,  383 

Meakins,  177,  370 

Mendel,  55 

Mennes.  257 

Metchnikoff,  43,  59,  60,  93,  107,  109, 
113,  125,  134,  137,  152,  166,  179, 
184,  190,  204,  238  et  seq.,  289,  293, 

335,  4°6 
Meyer,  411,  415 
Morax,  107 

Moreschi,  171,  172,  392 
Morgenroth,  45,  88,  177,  181 
Moro,  381 

Muir,  88,  163,  164,  282,  286,  341 
Miiller,  220,  417 
Myers,  82,  228,  234 


N 

Nefedieff,  193 
Neisser,  50,   70,    173,   217,   236,  323, 

324.  4J5 
Nernst,  87 
Netter,  317 

Neufeld,  257,  284,  285,  365 
Nicolle,  170,  209,  211,  219 
Nikayama,  292 
Nocard,  381 
Noguchi,  155,  232 
Norris,  227 
Norton,  395 
Nuttall,  139,  228,  232,  250 


Obermayer,  232,  234 
Osborne,  55 
Otto,  207,  312 

P 

Paget,  Sir  James,  361 
Pane,  366 
Panichi,  209 
Parascandalo,  360 
Park,  206,  220 
Parvu,  170 

Pasteur,  15,  -8,  19,  34 
Pauli,  298,  321,  324,  327 


Pearce,  191    193 

Perrin,  320 

Peskind,  223 

Petrie,  181 

Petterson,  182 

Pfeiffer,  58,  140,   162,   171,   184,  214, 

281,  392 
Pick,  232,  234 
Pierallini,  353 

Pirquet,  von,  303,  308,  315,  381 
Ponder,  296 
Porges,  417 
Posselt,  206 
Potzl,  417 
Pozerski,  170 

R 

Rankin,  372 
Ransom,  106,  411,  415 
Reagh,  215 
Reid,  282,  375 
Remy,  406 
Richet,  309 
Ricketts,  369 
Rimpau,  257,  365,  392 
Rist,  409 
Roger,  204,  221 
Rdmer,  106,  108,  351,  366 
Rosenau,  297,  312, 
Rosenfeld,  418 
Rosenow,  282,  291,  343 
Rossignol,  387 
Rostane,  317 
Roux,  86,  93,  414 
Rowland,  58 
Ruffer,  211,  214 
Ruppel,  373 

S 

Sachs,  154,  172,  236,  327 
Sagasser,  206 
Salmon,  25,  39 
Salmonsen,  93 
Sanarelli,  250 
Satchenko,  407 
Schattenfroh,  181 
Schereschewsky,  418 
Schick,  315 
Schmidt,  196 
Schutze,  228,  234 
Sclavo,  198,  407,  408 
Sellards,  295,  297 
Shattock,  262 
Shiga,  199,  397,  405 
Siedentopf,  319 
Simon,  263 
Smith,  25,  39,  215 
Smith,  Henderson,  286 
Smith,  Theobald,  311 
Sobernheim,  406,  408 
Soudakewitch,  336 
Southard,  312 


442 


INDEX    OF   AUTHORITIES    CITED 


Stern,  232,  233 
Stillmarck,  38,  55 
Stockman,  29 
Sturli,  224 

T 

Takaki,  106 
Tauber,  366 

Tchistovitch,  125,  227,  336 
Teague,  320,  329 
Tizzoni,  334 
Todd,  396 

Torrey,  215,  352,  368 
Tunnicliffe,  123,  262,  267,  270 


U 

Uhlenhuth,  228,  232,  235,  236 
Uschinsky,  54 


Vaillard,  86,  93,  113,  411 

Vallee,  387 

Van  de  Velde,  179 

Vincent,  113 

Volk,  216 

W 

Waage,  81 
Walker,  Ainley,  17,  183,  208,  219 


Walker,  R.,  262 
Washbourn,  364 

Wassermann,  44,  57,  71,  106,  184,  228, 
232,   235,   293,  303,   306,  337,  373, 

392,  4*5 

Wechsberg,  50,  70,  173,  323 
Weidenreich,  225 
Weichardt,  195 
Weigert,  98 
Weil,  291,  292 
Weinberg,  170 
Welch,  59,  219 
Western,  270,  395 
Whitfield,  278,  299 
Widal,  317 
Wiltshire,  223 
Woltmann,  194 
Wood,  Cartwright,  62,  65 
Wright,  Sir  A.,  25,  127,  189,  199  211, 

257  *  seq.,  333,  350,  360,  375,  4°2 


Yersin,  403 


Zammit,  375 
Ziemka,  235 
Zsigmondy,  319 


INDEX 


ABRIN,  54;  action  on  conjunctiva,  108 

Abscess,  cure  of,  349 

Absorption  of  complement.  See  Fixa- 
tion of  complement 

Acne,  358 

Acquired  immunity,  19 

Active  immunity,  20.     See  Glossary 

Addiment,  143.     See  Glossary 

Adsorption,  90,  392 

Age  in  relation  to  immunity,  8 

Agglutination  :  by  chemical  substances, 
211  ;  mechanism  of,  212;  salts  in, 
209,  326 

Agglutinins,  204  (see  Glossary)  ;  action 
of  heat  on,  205  ;  chemical  nature  of, 
214  ;  to  B.  diphtheria,  409  ;  to  B. 
dysenteria,  398  ;  effects  of  tempera- 
ture on,  208,  216  ;  formation  of,  208  ; 
in  normal  blood,  99  ;  to  gonococci, 
368  ;  mechanism  of  action,  212  ;  to 
meningococci,  372  ;  to  pneumococci, 
365  ;  relation  with  cytolysins,  207  ; 
role  in  immunity,  208  ;  sensitiveness 
of  bacteria  to,  219;  specificity  of,  205, 
217;  tostaphylococci,  359;  to  strepto- 
cocci, 360;  to  tubercle  bacilli,  383;  to 
typhoid  bacilli,  389 ;  to  V.  cholera,  400 

Agglutinogen,  210.     See  Glossary 

Agglutinoids,  47,  210,  216,  323.  See 
Glossary 

Aggressins,  291  (see  Glossary) ;  speci- 
ficity of,  292 

Air,  vitiated,  12 

Albumoses  in  bacterial  cultures,  54 

Alcohol,  13 

Alexins,  139  (see  Glossary)  ;  source  of, 
179 

Allergia,  308 

Amboceptor.  141  (see  Glossary)  ;  action 
as  opsonin,  285  ;  formation  of,  147  ; 
methods  of  investigating,  185;  source 
of,  184 

Amoeba,  phagocytosis  in,  238 

Anaesthesia.  12 


Anaphylaxis,  309.     See  Glossary 

Anaphylactin,  313 

Anthrax  bacilli :  immunity  to,  405  ; 
phagocytosis  of,  244,  250,  252  ; 
prophylaxis,  23,  407  ;  toxins  of,  405  ; 
treatment  of,  408 ;  vaccination 
against,  18,  23,  407 

Anti-abrin,  108 

Anti-agglutinin,  219 

Anti-aggressin,  292 

Anti-amboceptor,  160 

Anti-antibodies,  104 
j    Anti-autolysin,  150 

Antibodies,  site  of  production  of,  105,351 

Anticomplement,  157 

Anticrotin,  327 

And  enzymes,  48 

Anti-epithelial  serum,  197 

Antigen,  101.     See  Glossary 

Antihsemolysin,  109 

Anti-intestinal  serum,  195 

Antileucolysin,  50 

Antileucotoxin,  190 

Antilysin,  160 

Antispermotoxin,  109,  160 

Antistaphylolysin,  52,  358 

Antistreptocolysin,  360 

Antitoxin :  administration  by  mouth, 
131  ;  formation  of,  60,  97,  114;  in 
normal  blood,  93,  99  ;  production  by 
toxoids,  99  ;  reactions  with  toxin,  69  ; 
role  in  immunity,  119;  role  in  re- 
covery, 119  ;  unit  of,  72 

Antituberculin,  161,  307 

Arsenic,  absorption  by  leucocytes,  113 

Arthus'  phenomenon,  311.  See  Glossary 

Atoxyl  in  trypanosomiasis,  6,  16 

Atreptic  immunity,  35.     See  Glossary 

Atropin,  absorption  by  leucocytes,  113 

Aqueous  humour,  opsonin  in,  286 

Auto-agglutinin,  104,  222 

Auto-anticomplement,  158 

Autohsemolysin,  149.     See  Glossary 

Auto-inoculation,  268,  382 

Autonephrotoxin,  192 


443 


444 


INDEX 


B 

B.  anthracis.     See  Anthrax 
Bacillus  of  hotulismus,  toxin  of,  40 
B.    coli :    diseases   due    to,    393  ;    im- 
munity   to,   393  ;    toxins   of,    393  ; 

vaccine  treatment,  394 
B.  diphtheria.     See  Diphtheria 
Bacillus  of  dysentery,  toxins  of,  396 
B.  pyocyaneus  :  antagonism  to  anthrax, 

40  ;   antitoxin,    121  ;  hsemolysin    of, 

53  ;  leucolysin,  50 
B.  tetani.     See  Tetanus 
B.  typhosus.     See  Typhoid 
Bacteria,  immunity  to,  331 
Bacterial  hsemolysins,  40,  44 
Bactericidal  serum,  therapeutic  use  of, 

198 
Bactericidal   power  of  blood,   139  ;  of 

serum,  measurement  of,  188 
Bacteriolysis,  139.     See  Glossary 
Bacterio-precipitin,  226,  231 
Bacteriotropin.     See  Glossary 
Bazillen  emulsion,  384 
Bleeding    large    animals,    65 ;    small, 

185 

Blood,  human,  test  for,  233,  235 
Blood-relationship,  234 
Boils,  358 
Bone-marrow,  reaction  of,  in  infections, 

34i 
Bordet-Gengou  phenomenon,  153,  168. 

See  Glossary 

Bovine  tuberculosis,  diagnosis  of,  380 
Brain  substance  and  tetanus  toxin,  44, 

106 
Bright's  disease,  13,  193 


Calcium    chloride,    agglutination     by, 

211 

Calcium  lactate,  use  of,  317,  391 

Calmette's  test,  303,  382 

Capsules  (bacterial),  function  of,  343 

Carbuncles,  358 

Castellani's  absorption  reaction,  217 

Cayman,  reaction  to  tetanus  toxin,  60 

Cellulo-humoral  theory,  249 

Cerebro-spinal  fever,  371 

Cerebro-spinal  fluid,  373,  374 

Cervical  catarrh,  395 

Chemotaxis,  112,  244,  294,  341.  See 
Glossary 

Chicken  cholera,  aggressin  to,  291 

Cholecystitis,  395 

Cholera,  398;  bacteriolysis  in,  140,  337  ; 
diagnosis  of,  399  ;  endotoxin  of,  57, 
59  ;  Pfeiffer's  test  in,  140,  400  ;  pro- 
phylaxis, 400 ;  toxins,  398 

Cholesterin,  action  on  toxins,  107 


Coagulation    of    blood,    liberation 
complement  in,  182 

Coagulation  of  proteids,  321 

Cobra-lecithid,  156 

Cold  in  causation  of  disease,  9 

Colchicine,  latent  period  of,  41 

Colitis,  mucous,  395 

Colloidal  chemistry,  319 

Colloids,  90  ;   agglutination  of,  217 

Complement,  14.2,  145  (see  Glossary) ; 
as  opsonin,  285  ;  deviation  of,  I73> 
323;  (endo-),  156;  fixation  of,  170; 
methods  of  research  on,  185  ;  ori- 
gin of,  179,  252  ;  specificity  of, 
286 

Complementoid,  158.     See  Glossary 

Complementophile  haptophore  group, 
146 

Complementoids,  47.     See  Glossary 

Conjunctivitis,  368,  370 

Copula,  141 

Crisis  (in  pneumonia),  365 

Cuti-reaction,  303,  381 

Cystitis  (B.  coli},  394 

Cytase,  142,  167,  254.     See  Glossary 

Cytolysins,  190  et  seq.  (see  Glossary) ; 
bacterial,  40 

Cytophile  haptophore  group,  145 

Cytotoxin,  197 


1) 


Danysz  effect,  327.     See  Glossary 

Daphnia,  phagocytosis  in,  238 

Dead  bacteria  as  vaccines,  24 

Dendroccelum,  digestion  in,  241 

Desmon,  141.     See  Glossary 

Deuterotoxin,  75 

Deviation  of  complement,  173,  323. 
See  Glossary 

Diabetes,  13 

Digestion,  intracellular,  241 

Diphtheria  antitoxin  :  dosage  of,  410  ; 
in  normal  blood,  93 ;  standardiza- 
tion of,  45 

Diphtheria  bacillus,  antiserum  against, 
409 

Diphtheria  :  diagnosis  of,  409  ;  latency 
of,  33  ;  local  immunity  to,  30  ;  pro- 
phylaxis, 410 ;  toxin  of,  40  ;  action 
of,  49 ;  neutralization  of,  72,  85 ; 
standardization  of,  45 

Diphtheritic  paralysis,  72,  80,  87 

Dissociation,  28,  85 

Dominant  complement,  154.  See 
Glossary 

Dosage  of  vaccines,  24 

Dysentery,  396  ;  bacillus,  agglutination 
of,  220 ;  prophylaxis  of,  397  ;  treat- 
ment of,  397 


INDEX 


445 


Eclampsia,  cytolytic  theory  of,  195 

Eel  serum:  immunity  to,  125,  130,  135  ; 
precipitin  for,  228 

Ehrlich's  phenomenon,  328.  See  Glos- 
sary 

Electrolysis  of  toxins,  90,  92 

Endocomplement,  156.     See  Glossary 

Endothelial  cells  as  phagocytes,  246 

Endotoxin,  56,  339.     See  Glossary 

Enterokinase,  256 

Enzymes  :  analogies  with  toxins,  42  ; 
proteolytic,  in  pus,  337 

Epitoxoid,  76 

Epitoxonoid,  327 

Ergophore  group.     See  Glossary 

Erysipelas,  treatment  of,  362 

Evolution,  130,  165  ;  of  bacteria,  221, 

344 

Exhaustion,  Pasteur's  theory  of,  34 
Exotoxins,  48  (see  Glossary)  ;  chemical 

nature  of,  53 


False  rise,  274 

Fatigue,  10 

Fixation  of  complement,  153,  168,  236. 

See  Glossary 

Fixator,  141,  167.     See  Glossary 
Flagella,  agglutination  of,  227 
Food,  insufficient,  II 
Fowl  cholera,  18,  22 
Frog,  action  of  tetanus  toxin  on,  45 
Frontal  sinus  suppuration,  367 


Gastrotoxin,  194.     See  Glossary 

Gengou's  reaction.     See  Glossary 

Giant  cells,  378 

Gleet,  opsonic  index  in,  368 

Gonococci :  immunity  to,  369 ;  local 
immunity  to,  30,  369  ;  opsonic  index 
to,  368 ;  vaccines  in  disease  due  to, 
370 

Group  reactions,  217.     See  Glossary 

H 

Hsemagglutinin,  221.     See  Glossary 
Hsemolysins  :    bacterial,   40,    44,    50  ; 

serum,  141  et  seq. 
Haemolysis,    40,     141    (see   Glossary)  ; 

by   silicic    acid,    326 ;    methods    of 

research,  185 
Hsemolysoids,  46,  51 
Hsemopsonin,  245,  273,  285 
Haptines,  95.     See  Glossary 
Haptophore  group,  46.     See  Glossary 
Hepatotoxin,  192 
Heterolysins,  149 
Hog  cholera,  vaccination  against,  39 


Horse-flesh,  test  for,  237 
Horse-sickness,  vaccination  against,  28 
Hydatids,  diagnosis  of,  170 
Hypersensitiveness  to  toxins,  61,  121. 
See  Anaphylaxis 


Ichthyotoxin,  101,  125 

Immune  body,  141.     See  Glossary 

Immunisin,  141 

Immunitas  non  sterilisans,  16,  33,  331 

Immunity  :  acquired,  19  ;  active,  20  ; 
atreptic,  35  ;  bacterial,  331  ;  defini- 
tion of,  I ;  due  to  loss  of  receptors, 
125  ;  local,  29  ;  mixed,  28  ;  natural, 
7  ;  of  leucocytes,  128  ;  passive,  26  ; 
to  toxins,  115;  to  toxins,  natural, 

134 

Incitor  element.     See  Glossary 
Indol,  115 
Infection,  definition  of,  5  ;  predisposing 

causes  of,  9 
Interbody,  141 
Intermediary  body,  141 
Ions,  80 

Iritis,  gonococcal,  370 
Isoagglutinin,  221.     See  Glossary 
Isolysin,  149 
Isoprecipitin,  234 

K 

Koch's    phenomenon,    see   tuberculin. 

See  Glossary 
Kraus's  reaction,  209 


Latency  of  bacteria)  33  ;  of  tubercle 
bacilli,  387 

Latent  period  of  toxins,  41 

Lecithin  :  action  on  toxins,  107  ;  role 
in  haemolysis,  156,  180 

Lens,  crystalline,  precipitin  to,  233 

Lethal  dose,  minimal,  42 

Leucocytes  :  absorption  of  toxins  by, 
44,  113  ;  as  source  of  complement, 
179  ;  chemotactic  attraction  of,  112  ; 
degeneration  of,  122  ;  during  starva- 
tion, etc.,  12 ;  immunity  of,  128 ; 
in  combating  toxins,  120,  137  ;  in 
Metchnikoff  s  theory,  242  ;  prepara- 
tion of  emulsions  of,  257 

Leucocytosis  in  prognosis,  112,  341 

Leucolysins,  49,  358 

Leucopsenia,  341 

Leucotoxic  serum,  190 

Leucotoxins,  49,  358 

Liver,  phagocytosis  in,  336 

Local  lesion,  33  ;  cure  of,  346 

Local  immunity,  29,  125 

Lungs,  phagocytosis  in,  248,  336 


446 


INDEX 


M 

Macrocytase,  152,  254.     See  Glossary 

Macrophage,  247.     See  Glossary 

Malaria,  immunity  to,  33 

Mallein,  303 

Malta  fever,  374  ;  treatment  of,  375 

Meats,  recognition  of,  237 

Meningococcus,  toxins  of,  371  ;  phago- 
cytosis of,  371 

Meningitis :  serum  treatment,  373  ; 
vaccine  treatment,  374 

Micrococcus  melitensis,  agglutination  of, 

375 

Microcytase,  152,  254.     See  Glossary 
Microphage,  247.     See  Glossary 
Minimal  lethal  dose,  42 
Monospora,  phagocytosis  of,  239,  240 
Mytilo-congestine,  309 

N 

Nasik  vibrio,  toxin  of,  41 
Natural  immunity,  7 
Negative  phase,  62  (see  Glossary) ;    in 

opsonic  index,  274  ;  summation   of, 

276 
Neisser-Wechsberg  phenomenon,   173, 

323.     See  Glossary. 
Nephrotoxin,  192.     See  Glossary 
Nerves,  peripheral,  cytolytic  serum  for, 

196 

Neutralization  of  poison?,  116 
Neurotoxin,  196 
New  tuberculin,  384 
Nicotin,  absorption  by  liver,  116 
Nitrites,  production  of,  in  cholera,  37 
Nucleo-proteids  as  antigens,  192 


Ophthalmo-reaction,  303,  304,  382 

Ophthalmotoxic  serum,  197 

Opsonic  index,  261  ;  in  acute  diseases, 
265;  in  chronic  diseases,  268;  in 
diphtheria,  267  ;  effect  of  dilution  of 
serum,  264  ;  effect  of  vaccines,  274  ; 
in  erysipelas,  270  ;  false  rise  in,  274  ; 
to  gonococci,  270  ;  to  meningococci, 
371  ;  pre-agonal  rise  in,  280  ;  to 
pneumococci,  265,  361,  365  ;  in 
staphylococcic  diseases,  266 ;  tubercle 
bacilli,  268,  382 

"  Opsonins-therapy,"  277  ;  in  tubercle, 

385 

Opsonins:  etTect  of  temperature  on  their 
action,  295  ;  fundamental  experi- 
ments on,  257  ;  Metchnikoffs  views 
on,  289  ;  nature  of,  273 ;  origin  of, 
288  ;  presence  in  plasma,  286,  333  : 
specificity  of,  270 ;  thermostability, 
259,  282  ;  technique  of  researches  on, 
259-264;  thermolabile,  285  ;  thermo- 
stable, 259,  282 


Orthophosphoric  acid,  agglutination  by, 

216 
Osteomyelitis,  358 


Passage,  15,  219 

Passive  immunity,  26.     See  Glossary 

Peritoneum,  phagocytosis  in,  248,  250, 

352 

Perlsucht  tuberculin,  384 

Pfeiffer's  phenomenon,  141.  See  Glos- 
sary 

Phagocytic  index,  260 

Phagocytosis,  238  et  seq.;  action  of 
salts  in,  297;  in  circulating  blood, 
333  ;  in  peritoneum,  248,  250,  352  ; 
influence  of  source  of  leucocytes,  290  ; 
influence  of  temperature,  295  ;  influ- 
ence of  virulence,  343  ;  nature  of,  295 

Phagolysis,  181,  353 

Philocytase,  141 

Phthisis,  12 

Pigmentolysin,  197 

Piroplasma  bigeminum,  21 

Pirquet's  (von)  reaction,  303,  381 

Placentolysin,  195 

Plague,  402 ;  prophylaxis  of,  403  ; 
serum  treatment  of,  403 

Plasma,  complement  in,  182;  opsonin 
in,  286,  333 

Pleuralistic  conception,  151 

Pleuropneumonia  of  cattle,  22 

Pneumonia.     See  Pneumococci 

Pneumococci  :  agglutinins  to,  365 ; 
immunity  to,  365  ;  in  childhood,  8  ; 
opsonic  index  to,  266  ;  serum  treat- 
ment, 366  ;  vaccine  treatment,  366  ; 
virulence,  366 

Poisons  :  difference  from  toxins,  37  ; 
neutralization  of,  116 

Polyceptor.     See  Glossary 

Polyvalent  serum,  363  (see  Glossary)  ; 

vaccine  (see  Glossary) 
!    Positive  phase,  62.     See  Glossary 
:    Potato  bacillus,  phagocytosis  of,  248 
j    Precipitins,  226  (see  Glossary)  ;  speci- 
ficity of,  232,  235 

Precipitoid,  228,  323.     See  Glossary 

Precipitogenoid,  231.     See  Glossary 

Predisposing  causes,  9 

Preparator,  141.     See  Glossary 

Pro-agglutinin,  210 

Prostatotoxin,  196 

Proteids,  coagulation  of,  321 

Prototoxin,  75 

Prototoxoid,  73 

Pro-zones,  216,  323.     See  Glossary 

Pus,  enzymes  of,  337 

Pyocyaneus  (B. ) :  antagonism  to  anthrax, 
40  ;  toxin  of,  57 

Pyocyanolysin,  53 


INDEX 


447 


R 

Rabies,  vaccination  against,   23,   419  ; 

virus  of,  15 
Reactions  :    curative    effects    of,    281  ; 

tubercle,  etc.,  300 
Receptors,  95  (see  Glossary) ;  loss  of, 

I25.  343  ;  sessile,  126,  160  / 

Relapsing  fever,  recovery  from,  335  r 
Retention  theory.  Chauveau's,  35 
Reversible  reactions,  81 
Ricin,  38,  54,  55 

Rinderpest,  vaccination  against,  21 
Ringworm,  local  immunity  to,  30 


Salts,    role   of,    in   agglutination,  209, 

211 

Septicsemia,  332 

Serum  :  anti-anthrax,  408  ;  anticholera, 
400 ;  antidiphtheritic,  409 ;  anti- 
meningococcic,  373 ;  antipneumo- 
coccic,  366 ;  antiplague,  403  ;  anti- 
streptococcic,  363  ;  antitetanic,  413  ; 
antityphoid,  390-392  ;  bacteriolytic, 
use  of,  198;  disease,  315;  toxin, 
62 

Side-chains,  95 

Side-chain  theory,  94.    See  Glossary 

Smith's  (Theobald)  phenomenon,  311 

Specific  inhibition,  230 

Specificity,  19,  105  (see  Glossary)  ; 
of  agglutinins,  205,  217;  of  cyto- 
lysins,  191;  of  precipitins,  232 

Spectrum  of  toxin,  73 

Spermotoxin,  109,  190 

Spleen,  phagocytosis  in,  334 

Staphylococcus  pyogenes :  bacteriolysis, 
337;  leucolysin  of,  50,  358;  im- 
munity against,  359  ;  recovery,  347  ; 
toxins  of,  388 ;  vaccine  treatment  of, 

359 

Staphylolysin,  51,  52,  358 
Starvation,  II 
Sdmulins,  122,  295 
Stomach,  immunity  of,  29 
Streptococci :  immunity  to,  360  ;  serum 

treatment   of   disease    due    to,  362  ; 

toxins  of,  359 ;  vaccine  treatment  of 

disease  due  to,  362 
Streptococcus  pyogenes  :  hcemolysin  of, 

5°)  359  I  leucolysin  of,  50 
Streptocolysin,  50 
Substance  sensibilatrice^  141 
Surface-tension,  213,  298 
Swine  erysipelas,  19,  22 
Symbiosis  of  leucocytes,  248 
Sympathetic  ophthalmia,  197 
Syncytiolysin,  195 
Syphilis,  415  ;  Wassermann's  reaction 

in,  415 


;    Teianolysin,  52,  83,  411 

Tetanospasmin,  52,  411 

Tetanus:  diagnosis  of,  410;  immunity 
to,  412  ;  passive   immunity   to,    27  ; 
prophylaxis  of,   413;    treatment  of, 
414  ;  toxin,  40,  47,  411  ;  absorption 
of,  by  brain,  44,  106  ;  leucocytes,  44; 
tissues,   44;  action  of,  49,   116;   on 
various  animals,    133  ;    antitoxin  to, 
413  ;  effect  of  temperature  on,  45 
i    Texas  fever,  vaccination  against,  21 
!    Thyrotoxin,  197.     See  Glossary 

Tick  fever,  335 

Tissue  immunity.    See  Local  immunity 

Toxalbumin,  54 

Toxins :  action  of,  41  ;  composition  of, 
47  ;  electrolysis  of,  90,  92  ;  hyper- 
sensitiveness  to,  61,  309;  immunity 
to,  115  ;  spectrum,  73  ;  standardi- 
zation of,  45,  71  ;  union  with  tissues, 
100 

Toxoids,  46,  47,  80  (see  Glossary) ;  pro- 
duction of  antitoxin  by,  99  ;  use  in 
immunization,  62 

Toxone,  73,  80.     See  Glossary 

Toxonoid,  88 

Toxophore  group,  46.     See  Glossary 

Trichina  spiralis,  29 

Trichotoxin,  192.    See  Glossary 

Tritotoxin,  76 

Trypanosomiasis,  6,  1 6 

Tubercle  bacillus  :  antibodies  for,  337  ; 
immunity  to,  377  ;  opsonic  index 
to,  377  ;  phagocytosis  of,  251,  377  ; 
toxins  of,  314,  376  ;  toxins  of,  Mar- 
morek's,  383 

Tuberculin  :  dilution  of,  380  ;  immuni- 
zation to,  303  ;  old,  300,  379 ;  re- 
action, 301,  378  ;  theories  of  reaction, 
305,  306,  308  ;  reaction  in  cattle, 
380  ;  R,  383  ;  therapeutic  use  of, 

384 

Tuberculosis  :  diagnosis  of,  379 ;  op- 
sonin  therapy  of,  385  ;  prophylaxis 
of,  386  ;  prophylaxis  in  cattle,  387 

Tulase,  387 

Typhoid  bacillus :  agglutinins  to,  206, 
210,  215 ;  agglutinins  in  normal 
blood,  99  ;  endotoxin  of,  53  ;  haemo- 
lysin  of,  53 ;  immunity,  388  ;  in 
peritoneum,  353 ;  latency  of,  33  ; 
phagocytosis  of,  263,  389  j  virulence 
of,  17,  343 

Typhoid  fever  :  bacterisemia  in,  332  ; 
bacteriolysis  in,  337 ;  opthalmo-re- 
action  in,  304  ;  prophylaxis  of,  28, 
390 

Typholysin,  53 

Tyrosin  :  action  on  toxins,  107 


448 


INDEX 


U 

Ulcerative  endocarditis,  332 
Ulcus  serpens,  treatment. of,  366 
Unitarian  theory  of  complement,  152 
Unit  of  toxin,  72  ;  of  antitoxin,  72 
Uraemia,  cytolytic  theory  of,  192 


Vaccine  treatment :  for  gonococcic 
diseases,  370  ;  for  Malta  fever,  375  ; 
for  meningitis,  374 ;  for  pneumo- 
coccic  diseases,  366 ;  for  staphylo- 
coccic  diseases,  359  ;  for  strepto- 
coccic  diseases.  362  ;  theory  of,  276  ; 
tubercle  (see  Tuberculin) 

Vaccines  :  chemical,  25,  39  ;  of  dead 
bacteria,  24  ;  dosage  of,  24  ;  method 
of  preparation,  1 8 

Vaccinia,  22 

Venom  (snake), '155 

Vibrio,  Nasik,  41 


Vibrio    Metchnikovi,    bacteriolysis    of, 

173 

Vibrio  Nordhafen,  bacteriolysis  of,  174 
Virulence,  14  ;  capsule  formation, 
effect  of,  on,  343  ;  changes  in,  15,  17  ; 
diminution  in,  methods  of  produc- 
tion, 18  ;  increase  in,  methods  of 
production,  15,  17,  219;  influence 
on  phagocytosis,  295  ;  mechanism 
of,  343 

Virus  (see  Glossary),  22  ;  fixed,  15  ;  of 
the  streets,  15 

W 

Wassermann's  reaction,  416 
Wet,  in  causation  of  disease.  9 
Widal  reaction,  389 


Zones  of  inhibition,  216 

Zoological  affinities  :  immunity  in  re- 
lation to,  8  ;  relations  to  precipitins, 
234 


K.  LEWIS,  136,  GOWER  STREET,  LONDON,  W.C. 


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